Dysosmobacter, a novel bacterial genus of the gastrointestinal microbiota and uses thereof

ABSTRACT

A bacterium belonging to a novel bacterial genus, Dysosmobacter. Also, the therapeutic, nutraceutical and cosmetic use thereof. The uses include methods for treating a disorder, promoting weight loss, decreasing food intake, increasing muscle mass, decreasing fat mass, increasing satiety, and/or decreasing weight gain associated with food intake in a subject, including administering to the subject at least one isolated bacterium belonging to the genus Dysosmobacter and/or a variant thereof and/or fragments thereof.

FIELD OF INVENTION

The present invention relates to the gut microbiota. More specificallythe present invention relates to bacteria of the genus Dysosmobacter andtheir use in the treatment of gut microbiota-related disorders.

BACKGROUND OF INVENTION

The human gut is colonized by a diverse, complex and dynamic communityof microbes representing over 1000 different species, which continuouslyinteract with the host. The homeostasis of the gut microbiota isdependent on host characteristics (age, gender, genetic background . . .) and environmental conditions (stress, drugs, gastrointestinal surgery,infectious and toxic agents . . . ), but also on the day-to-day dietarychanges.

It has been recently acknowledged that the intestinal microbiota isinvolved in a number of diseases. For instance, gut microbiota imbalancewas shown to be a risk factor for the development of cancers such ascolorectal cancer. Growing evidences also support the role of gutmicrobiota in the development of obesity and related disorders, braindisorders and intestinal inflammation, or intestinal pain.

Therefore, treatment with products that target the gut microbiotaappeared as promising therapeutic tools for treating a broad range ofdisorders. These products may consist of living microbes, such as in thecase of most probiotics, or contain dead microbes or fragments thereof.In addition, these products may comprise substrates that are used by thegut microbiota, such as in the case of prebiotics, or contain compoundsthat change the balance of the intestinal microbiota, such as specificantimicrobial compounds. For example, WO 2008/076696 describes the gutmicrobiota as a therapeutic target for treating obesity and relateddisorders. WO 2008/076696 specifically describes methods for alteringthe abundance of Bacteroidetes and/or Firmicutes in the gut of asubject, by administering antibiotics and/or probiotics to the subject.Moreover, EP 2 030 623 relates to the prevention and/or treatment ofmetabolic disorders, such as, for example, obesity related disorders, byregulating the amount of Enterobacteria in the gut. EP 2 030 623discloses reducing the amount of Enterobacteria in the gut byadministering probiotic bacteria, such as, for example, Bifidobacterium,Lactococcus, Streptococcus, Enterococcus or Lactobacillus.

However, there is still a need for other products, for example derivedfrom the gut microbiota, with a therapeutic potential for treatingmicrobiota related diseases.

Clostridial cluster IV is a phenotypically heterogeneous group thatincludes motile as well as non-motile species, sporulating as well asnon-sporulating bacteria and Gram-staining positive, negative orvariable species. The majority of them are anaerobic rods isolated fromor at least encountered in the digestive tract of various animals, forexample corbicula clams with Oscillibacter valericigenes, cattle withOscillibacter ruminantium, wood-feeding termite with Sporobactertermitidis, cat with Agathobaculum desmolans and human with Clostridiumleptum, Faecalibacterium prausnitzii or Papillibacter cinnamivorans.Several of them, F. prausnitzii and Butyricicoccus pullicaecorum inparticular, are major butyrate producers in the gastrointestinal tractand have demonstrated health-promoting properties (Sokol et al., ProcNatl Acad Sci USA. 2008 Oct. 28; 105(43):16731-6; Eeckhaut et al., Gut.2013 December; 62(12):1745-52). Others are suspected to have beneficialproperties due to their regular association with health-relatedparameters in cultivation-independent studies. This is the case ofOscillospira guilliermondii, which is positively associated withleanness and negatively associated with inflammatory bowel disease andliver disease (Konikoff et al., Trends Microbiol. 2016 July;24(7):523-524).

The absence of cultivable isolated strains representative of those taxahinders a better understanding of the physiology of those bacteria andthe demonstration of their causal impact on animal health.

In the present invention, the applicants were able to isolate andcultivate a novel bacterial strain, J115, from a fecal sample of ahealthy 25 years old female, in an effort to identify potential newbeneficial microbes isolated from the human gut. Strain J115 representsthe type strain of a novel species, Dysosmobacter welbionis, itself thetype species of a new genus, Dysosmobacter, whose identification hasopened the way for its use in the treatment of gastrointestinalmicrobiota related disorders. Surprisingly, the applicants hereindemonstrated that this novel bacterial species, when administered invivo, presents a therapeutic potential for treating diseases related tothe gut microbiota, as well as a cosmetic potential, such as, forexample, for inducing weight loss.

The present invention thus relates to bacterial cells of theDysosmobacter genus, and to the use thereof in therapeutic or cosmeticmethods.

SUMMARY

The present invention relates to an isolated bacterium belonging to thegenus Dysosmobacter and/or a variant thereof and/or fragments thereof.

In one embodiment, the bacterium according to the invention belongs tothe species Dysosmobacter welbionis and/or a variant thereof.

In one embodiment, the nucleotide sequence of the 16S rRNA gene of theisolated bacterium of the invention has at least about 90% identity withSEQ ID NO: 1.

In one embodiment, the isolated bacterium according to the invention isable to ferment myo-inositol.

In one embodiment, the isolated bacterium according to the invention isthe strain J115, deposited at the BCCM/LMG on Mar. 14, 2018 as LMGP-30603, and/or a variant thereof.

In one embodiment, the isolated bacterium according to the invention ispasteurized.

In one embodiment, the isolated bacterium according to the invention isfrozen.

The present invention also relates to a composition comprising anisolated bacterium according to the invention, and/or fragments thereof.

The present invention also relates to a pharmaceutical compositioncomprising the composition according to the invention, and at least onepharmaceutically acceptable excipient.

The present invention also relates to a nutraceutical compositioncomprising the composition according to the invention, and at least onenutraceutically acceptable excipient.

The present invention also relates to a cosmetic composition comprisingthe composition according to the invention and at least one cosmeticallyacceptable excipient.

The present invention also relates to the isolated bacterium, thecomposition or the pharmaceutical composition according the invention,for use as a medicament.

The present invention also relates to the isolated bacterium, thecomposition or the pharmaceutical composition according the invention,for use in treating a disorder related to the gastrointestinalmicrobiota in a subject in need thereof.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a metabolic disease, preferably selected from the listcomprising obesity, metabolic syndrome, insulin-deficiency orinsulin-resistance related disorders, Diabetes Mellitus, glucoseintolerance, abnormal lipid metabolism, hyperglycemia, dyslipidemia,high cholesterol, elevated LDL-cholesterol, decreased HDL-cholesteroland elevated triglycerides

The present invention also relates to the use of the isolated bacterium,the composition, the nutraceutical composition or the cosmeticcomposition according to the invention, for promoting weight loss,decreasing food intake, increasing muscle mass, decreasing fat mass,increasing satiety, and/or decreasing weight gain associated with foodintake in a subject.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   “About” preceding a figure means plus or less 10% of the value        of said figure.    -   “Acceptable”, for example when used in the expressions        “Pharmaceutically acceptable”, “Nutraceutically acceptable” and        “Cosmetically acceptable” refers to molecular entities and        compositions that do not produce an adverse, allergic or other        untoward reaction when administered to a subject, especially a        human, as appropriate.    -   “Bacterial strain” refers to a subtype of a bacterial species.    -   “Clostridium cluster IV” refers to a phenotypically        heterogeneous group that includes motile as well as non-motile        species, sporulating as well as non-sporulating bacteria and

Gram-staining positive, negative or variable species. The majority ofthem are anaerobic rods isolated from or at least encountered in thedigestive tract of various animals, for example corbicula clams withOscillibacter valericigenes, cattle with Oscillibacter ruminantium,wood-feeding termite with Sporobacter termitidis, cat with Agathobaculumdesmolans and human with Clostridium leptum, Faecalibacteriumprausnitzii or Papillibacter cinnamivorans. Several of them, F.prausnitzii and Butyricicoccus pullicaecorum in particular, are majorbutyrate producers in the gastrointestinal tract and have demonstratedhealth-promoting properties. Others are suspected to have beneficialproperties due to their regular association with health-relatedparameters in cultivation-independent studies. This is the case ofOscillospira guilliermondii, which is positively associated withleanness and negatively associated with inflammatory bowel disease andliver disease.

-   -   “Cosmetically effective amount” refers to the amount of a        cosmetic composition necessary and sufficient for promoting a        cosmetic effect, such as, for example, for inducing weight loss        in a subject.    -   “Dusodibacter” [dy.so.di.bac'ter. a Gr. n. dusôdía putrescent,        fetid smell; N.L. masc. n. bacter rod; N.L. masc. n. a rod        producing a fetid smell] and “Dysosmobacter” [Dys.os.mo.bac'ter.        Gr. masc. adj. dysosmos bad smelling; N.L. masc. n. bacter a        rod; N.L. masc. n. Dysosmobacter a bad-smelling rod] are used        interchangeably to refers to a new genus of bacteria described        herein that have the following properties: cells are obligatory        anaerobic, non-pigmented, non-spore-forming, non-motile,        Gram-stain-negative. Cells form straight rods mainly 1.8-3.0 μm        but often form elongated rods up to 20 μm whatever the growing        phase. No respiratory menaquinones are produced. The genus        belongs to the family of Ruminococcaceae. The type species is        Dysosmobacter welbionis. In one embodiment, the diagnostic        diamino acid in the cell wall is meso-2,6-diaminopimelic acid.    -   “Dusodibacter welbiota” [u'εl.bi'o.ta. welbiota] and        “Dysosmobacter welbionis” [wel.bi.o'nis. N.L. gen. n. welbionis]        are used interchangeably to refer to a new species of bacteria        described herein that have the following properties in addition        to the properties of the genus Dysosmobacter described        hereinabove: colonies on solid modified YCFA after 72 h of        incubation at 37° C. under anaerobic conditions are punctiform,        cream, translucent, circular, entire, slightly convex and        smooth. Growth is inhibited by the presence of 2% w/v bile or 2%        w/v NaCl. Aesculin is not hydrolysed. Indole is not produced.        Nitrate is not reduced. Gelatin is not digested. Urease is not        produced. Catalase is not produced. Acid is produced from        myo-inositol but not from D-glucose, D-arabinose, D-ribose and        D-xylose. Positive reactions are obtained for arginine        dihydrolase and glutamic acid decarboxylase. All the other tests        from API 20A and Rapid ID 32A (bioMérieux, Lyon, France) are        negative. Major fermentation end-products from myo-inositol are        butyrate. The DNA GC content of the type strain is 59.3 mol % by        High Performance Liquid Chromatography (HLPC). In one        embodiment, the DNA GC content of the type strain is 58.9 mol %        on the basis the genomic sequence. Type strain is J115        (deposited at the BCCM/LMG on Mar. 14, 2018 as LMG P-30603) and        was isolated from human faeces. In one embodiment, the major        cellular fatty acids are saturated branched-chain fatty acids        and DMAs. In one embodiment, the major DMA fatty acid is        C_(18:0) DMA and major saturated branched-chain fatty acids are        iso-C_(15:0) and anteiso-C_(15:0).    -   “Fermentation” refers to the metabolic process that consumes        sugar in the absence of oxygen. The products are organic acids,        gases, or alcohol. It occurs in yeast and bacteria, and also in        oxygen-starved muscle cells, as in the case of lactic acid        fermentation.    -   “Gastrointestinal microbiota related disorder” or        “gastrointestinal microbiota related disease” are used        interchangeably to refer to a group of diseases or disorders        that are related to an imbalance, a deficiency or an excess in        the composition of a subject gastrointestinal microbiota and/or        product thereof. Example of disorders related to the        gastrointestinal microbiota include, but are not limited to        metabolic diseases such as for example, obesity, metabolic        syndrome, insulin-deficiency or insulin-resistance related        disorders, Diabetes Mellitus (such as, for example, Type 2        Diabetes), glucose intolerance, hyperglycemia, abnormal lipid        metabolism, dyslipidemia, high cholesterol, elevated        LDL-cholesterol, decreased LDL cholesterol and elevated        triglycerides, infections, colitis, such as for example,        inflammatory bowel disease (e.g., Crohn's disease and ulcerative        colitis), ischemic colitis, irritable bowel syndrome,        lymphocytic colitis and collagenous colitis, cancers, such as        for example, colorectal cancer, dysfunction of the immune        system, such as for example, eczema, allergies, food allergies        and celiac disease, psychological disorders, such as for        example, stress, anxiety and addiction, neurological disorders,        such as for example, Parkinson's disease and Alzheimer's        disease, liver diseases, such as for example, cirrhosis,        non-alcoholic fatty liver disease, and hepatic steatosis,        cachexia, prader-willy syndrome, dysfunction of the digestive        tract, such as for example, ulcers and gallbladder disease,        feeding behaviors disorders such as for example, anorexia        nervosa, bulimia nervosa and binge-eating disorder,        cardiovascular diseases and conditions, such as for example        strokes, atherosclerosis and hypertension, asthma, sleep apnea        and osteoarthritis.    -   “Gut microbiota” or “gastrointestinal microbiota” are used        interchangeably to refer to the complex community of        microorganisms that live in the digestive tracts of humans and        other animals. The composition of the gastrointestinal        microbiota changes over the lifetime of the host organism or        when the diet of the host changes. It also varies across the        digestive tract. The digestive tract contains a        densely-populated microbial ecosystem with up to 10¹² cells per        gram of intestinal content. Many species in the gut have not        been studied outside of their hosts because most cannot be        cultured. The four dominant bacterial phyla in the human gut are        Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria.        Most bacteria belong to the genera Bacteroides, Clostridium,        Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus,        Peptostreptococcus, Blautia, Subdoligranulum, Alistipes,        Coprococcus, Dialister, Lachnoclostridium, Oscillospira,        Parabacteroides, Prevotella, Roseburia, Ruminiclostridium,        Sutterella and Bifidobacterium. Other genera, such as        Escherichia, Enterococcus, Barnesiella, Butyricimonas,        Butyricicoccus, Lachnospira, Odoribacter, Turicibacter and        Lactobacillus, are present to a lesser extent. The gut        microbiota is thought to function in the defense again        pathogens, by competing with potential pathogens and by        participating in the development of enteric protection and the        immune system, metabolism, by assisting the digestion of        consumed aliments, aiding the absorption of nutrients and        synthetizing vitamins. The gut microbiota also interacts with        the function of the central nervous system and the        neuroendocrine and neuroimmune systems.    -   “Menaquinones” refers to components of the bacterial respiratory        chain that play an important role in electron transfer during        microbial respiration.    -   “Mutant”, as used herein, refers to a biological entity which        has undergone a natural or induced (i.e. by mutagenesis) change        in its genetic structure that does not interfere with the        defining properties of said biological entity. The change in its        genetic structure may be an insertion or deletion or        substitution of one or several nucleotides in the genomic        sequence. For example, a Dysosmobacter welbionis mutant refers        to a Dysosmobacter welbionis strain which has undergone a        change, natural or by techniques of genetic engineering, in its        genetic structure that does not interfere with its belonging to        the Dysosmobacter welbionis species.    -   “Nutraceutically effective amount” refers to the amount of a        nutraceutical composition, food or dietary supplement or        functional food necessary and sufficient for providing a        physiological benefit or alleviating a discomfort in a subject.    -   “Pasteurized bacterium” refers to a bacterium submitted to a        heat treatment (or heating process).    -   “Pharmaceutically acceptable carrier or excipient” refers to        molecular entities and compositions that do not produce an        adverse, allergic or other untoward reaction when administered        to a subject, especially a human, as appropriate. It includes        any and all solvents, dispersion media, coatings, antibacterial        and antifungal agents, isotonic and absorption delaying agents        and the like. For human administration, preparations should        meet, pyrogenicity, general safety and purity standards as        required by regulatory offices, such as, for example, FDA Office        or EMA. A pharmaceutically acceptable carrier or excipient may        thus refer to a non-toxic solid, semi-solid or liquid filler,        diluent, encapsulating material or formulation auxiliary of any        type.    -   “Prebiotic” refers to a substance, which may not be digested by        a subject (such as, for example, by humans), but which modulates        composition and/or activity of the gut microbiota through its        metabolization by microorganisms in the gut, thus conferring a        beneficial physiological effect on the host.    -   “Probiotics” refers to microbial cell preparations (such as, for        example, living microbial cells) which, when administered in an        effective amount, provide a beneficial effect on the health or        well-being of a subject. By definition, all probiotics have a        proven non-pathogenic character. In one embodiment, these health        benefits are associated with improving the balance of human or        animal microbiota in the gastro-intestinal tract, and/or        restoring normal microbiota.    -   “Subject” refers to a warm-blooded animal, preferably a human, a        pet or livestock. As used herein, the terms “pet” and        “livestock” include, but are not limited to, dogs, cats, guinea        pigs, rabbits, pigs, cattle, sheep, goats, horses and poultry.        In some embodiments, the subject is a male or female subject. In        some embodiments, the subject is an adult or a child. In some        embodiments, the subject may be a “patient”, i.e., a subject        who/which is awaiting the receipt of or is receiving medical        care or was/is/will be the object of a medical procedure        according to the methods of the present invention or is        monitored for the development of a disease.    -   “Substantially healthy subject” is used to define a subject        which is not affected by the disease to be treated or by the        discomfort to be alleviated. For example, if the bacterium of        the invention or a fragment thereof is used for treating        obesity, the substantially healthy subject is not affected by        obesity. Preferably, the substantially healthy subject shares        common characteristics with the subject to be treated, such as,        for example, same gender, age, sex, diet, drugs intake or        geolocation.    -   “Therapeutically effective amount” refers to the level or amount        of an agent that is aimed at, without causing significant        negative or adverse side effects to the target, (1) delaying or        preventing the onset of a disease, disorder, or condition; (2)        slowing down or stopping the progression, aggravation, or        deterioration of one or more symptoms of the disease, disorder,        or condition; (3) bringing about ameliorations of the symptoms        of the disease, disorder, or condition; (4) reducing the        severity or incidence of the disease, disorder, or condition;        or (5) curing the disease, disorder, or condition. A        therapeutically effective amount may be administered prior to        the onset of the disease, disorder, or condition, for a        prophylactic or preventive action. Alternatively, or        additionally, the therapeutically effective amount may be        administered after initiation of the disease, disorder, or        condition, for a therapeutic action.    -   “Treatment” refers to both therapeutic treatment and        prophylactic or preventative measures wherein the object is to        prevent or slow down (lessen) the targeted pathologic condition        or disorder. Those in need of treatment include those already        with the disorder as well as those prone to have the disorder or        those in whom the disorder is to be prevented. A subject or        mammal is successfully “treated” if, after receiving a        therapeutic amount of Dysosmobacter welbionis and/or a variant        thereof and/or a fragment thereof according to the present        invention the patient shows one or more of the following        observable and/or measurable changes: amelioration related to        one or more of the symptoms associated with the specific disease        or condition, reduction of morbidity and mortality and        improvement in quality of life issues. The above parameters for        assessing successful treatment and improvement in the disease        are readily measurable by routine procedures familiar to a        physician.    -   “Type strain” refers to as defined in the International Code of        Nomenclature of Bacteria, as the nomenclatural type of the        species and the reference point to which all other strains are        compared to know whether they belong to that species. For        example, strain J115, isolated from a faecal sample of a healthy        25 years old female, is the type strain of the species        Dysosmobacter welbionis.    -   “Variant” refers to all the genetically or phenotypically        distinct strains of a species that retain the species-defining        characteristics. The term variant is also used in reference to        other phylogenetic taxa such as for a genus or for a strain. As        used herein, the term “variant” refers to both naturally        occurring and specifically developed variants or mutants of the        bacterium disclosed and exemplified herein. In one embodiment,        variants may or may not have the same identifying biological        characteristics of the bacterium exemplified herein, provided        they share similar advantageous properties in terms of treating        or preventing diseases. In one embodiment, a variant of the        bacterium of the invention has the same functional and/or        therapeutic properties as the bacterium of the invention.        Illustrative examples of suitable methods for preparing variants        of the microbial strains exemplified herein include, but are not        limited to, gene integration techniques such as those mediated        by insertion of elements or transposons or by homologous        recombination, other recombinant DNA techniques for modifying,        inserting, deleting, activating or silencing genes,        intraspecific protoplast fusion, mutagenesis by irradiation with        ultraviolet light or X-rays, or by treatment with a chemical        mutagen such as nitrosoguanidine, methyl methane sulfonate,        nitrogen mustard and the like, and bacteriophage-mediated        transduction. Suitable and applicable methods are well known in        the art and are described, for example, in J. H. Miller,        Experiments in Molecular Genetics, Cold Spring Harbor Laboratory        Press, Cold Spring Harbor, N.Y, (1972); J, H. Miller, A Short        Course in Bacterial Genetics, Cold Spring Harbor Laboratory        Press, Cold Spring Harbor, N.Y. (1992); and J. Sambrook, D.        Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold        Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.        (2001)» inter alia.

DETAILED DESCRIPTION

The present invention first relates to a bacterium (or to a bacterialcell) belonging to the genus Dysosmobacter. Following a nomenclaturechange, the genus is also known as Dysosmobacter. Both terms areequivalent and interchangeable throughout the present application.Should the taxonomy change again, the skilled artisan would know how toadapt the changes in the taxonomy to deduce the bacteria that could beused in the present invention.

Bacteria belonging to the genus Dysosmobacter are described for thefirst time herein by the Inventors. The genus Dysosmobacter belongs tothe family Ruminococcaceae. In one embodiment, bacteria belonging to thegenus Dysosmobacter have the following characteristics: cells areanaerobic; not pigmented; non-spore-forming; non-motile, Gramstain-negative; cells form rods, e.g., straight rods (such as, forexample, about 1.8 to 3 μm long) or elongated rods; cells do not producerespiratory menaquinones; at least about 10%, preferably at least about20%, more preferably about 30% of cellular fatty acids are saturatedbranched-chain fatty acids and DMA fatty acid; C_(18:0) DMA representsat least about 5%, preferably at least about 10%, more preferably about15% of cellular fatty acids; iso-C_(15:0) represents at least about 10%,preferably at least about 15%, more preferably about 20% of cellularfatty acids; and anteiso-C_(15:0) represent at least about 5%,preferably at least about 7.5%, more preferably about 10% of cellularfatty acids. In one embodiment, the diagnostic diamino acid in the cellwall of bacteria belonging to the genus Dysosmobacter ismeso-2,6-diaminopimelic acid.

The sequence of the 16S rRNA gene of Dysosmobacter welbionis strainJ115, SEQ ID NO: 1 has been deposited under the GenBank/EMBL/DDBJaccession number MG963288.

The sequence of the 16S rRNA gene is often used to identify differentbacterial species because of the mutation accumulating in itshyper-variable regions and the presence of the gene in all bacteria.

In one embodiment, the nucleotide sequence of the 16S rRNA gene of thebacterium of the invention has the sequence SEQ ID NO: 1, or has asequence presenting at least about 90% identity with SEQ ID NO: 1,preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%,97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%,98.5%, 98.6%, 98.65%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identity with SEQ IDNO: 1.

In one embodiment, the nucleotide sequence of the 16S rRNA gene of thebacterium of the invention has the sequence SEQ ID NO: 1, or has asequence presenting at least about 99.9% identity with SEQ ID NO: 1,preferably at least about 99.91%, 99.92%, 99.93%, 99.94%, 99.95%,99.96%, 99.97%, 99.98%, 99.99%, or more identity with SEQ ID NO: 1.

In one embodiment, the nucleotide sequence of the 16S rRNA gene of thebacterium of the invention has the sequence SEQ ID NO: 1, or has asequence presenting at least about 90% identity or more identity overthe entire length of SEQ ID NO: 1, preferably at least about 91%, 92%,93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%,98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, 98.7%, 98.8%,98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%,or more identity over the entire length of SEQ ID NO: 1.

The term “identity” when used in a relationship between the sequences oftwo or more polypeptides or of two or more nucleic acid molecules,refers to the degree of sequence relatedness between polypeptides ornucleic acid molecules, as determined by the number of matches betweenstrings of two or more amino acid or nucleotide residues. “Identity”measures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”) Identity ofrelated polypeptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods fordetermining identity are designed to give the largest match between thesequences tested. Methods of determining identity are described inpublicly available computer programs. Preferred computer program methodsfor determining identity between two sequences include the GCG programpackage, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387(1984); Genetics Computer Group, University of Wisconsin, Madison,Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. MoI. Biol. 215,403-410 (1990)). The BLASTX program is publicly available from theNational Center for Biotechnology Information (NCBI) and other sources(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschulet al., supra). The well-known Smith Waterman algorithm may also be usedto determine identity. In one embodiment, the term identity is measuredover the entire length of the sequence to which it refers.

In one embodiment, the bacterium of the invention belongs to the speciesDysosmobacter welbionis. Following a nomenclature change, the species isalso known as Dusodibacter welbiota. Both terms are equivalent andinterchangeable throughout the present application.

In one embodiment, bacteria belonging to the species Dysosmobacterwelbionis, described for the first time herein by the Inventors, have,in addition to the characteristic described hereinabove of the genusDysosmobacter, the following characteristics: colonies forming on solidmodified YCFA medium after 72 h of incubation at 37° C. under anaerobicconditions are punctiform, cream, translucent, circular, entire,slightly convex and smooth. In one embodiment, bacterial growth isinhibited by a concentration of bile in the medium at or above about 1%w/v, preferably about 2% w/v and/or by a concentration of NaCl in themedium at or above about 1% w/v, preferably about 2% w/v. In oneembodiment, cells do not have catalase activity. In one embodiment,cells have arginine dihydrolase and/or glutamic acid decarboxylaseactivity.

In one embodiment, the bacterium of the invention is able to fermentmyo-inositol.

In one embodiment, the bacterium of the invention is unable to fermentD-glucose, D-ribose, D-arabinose and D-Xylose.

In one embodiment, the bacterium of the invention is unable to fermentD-glucose and/or D-Xylose.

Techniques to determine the substrates that a bacterium is able toferment are known to the person skilled in the art. For example, thischaracterization can be done using anaerobe test kit such as the testAPI 50CH (BioMérieux, Lyon, France).

In one embodiment, at least 1, preferably at least 2, 3, 4, 5, 6, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 tests from the anaerobetest kit API20A (BioMérieux, Lyon, France) are negative.

The anaerobe test kit API20A (BioMérieux, Lyon, France) allows thebiochemical characterization of anaerobe bacteria by enabling 20biochemical tests, namely urease activity, indole production, gelatinhydrolysis, esculin hydrolysis, fermentation of D-glucose, D-mannitol,D-lactose, D-saccharose, D-maltose, salicin, D-xylose, L-arabinose,glycerol, D-cellobiose, D-mannose, D-melezitose, D-raffinose,D-sorbitol, L-rhamnose and D-trehalose.

In one embodiment, at least 1, preferably at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 or 32 tests from the anaerobe test kit API32A(BioMérieux, Lyon, France) are negative.

The rapid ID32A (BioMérieux, Lyon, France) allows the biochemicalcharacterization of anaerobe bacteria by enabling 32 biochemical tests,namely urease, arginine dihydrolase, α-galactosidase, β-galactosidase,β-galactosidase-6-phosphate, α-glucosidase, α-arabinosidase,β-glucuronidase, N-acethyl-glucosaminidase, glutamic acid decarboxylase,α-fucosidase, alkaline phosphatase, arginine arylamidase, prolinearylamidase, leucyl glycine arylamidase, phenylalanine arylamidase,leucine arylamidase, pyroglutamic acid arylamidase, tyrosinearylamidase, alanine arylamidase, glycide arylamidase, histidinearylamidase, glutamic acid glutamyl arylamidase and serine arylamidaseenzymatic activities as well as nitrate reduction, indole production andmannose and raffinose fermentation.

In one embodiment, the bacterium of the invention is not motile. In oneembodiment, the bacterium of the invention has no flagellum.

In one embodiment, the bacterium of the invention is the strain J115(deposited at the BCCM/LMG on Mar. 14, 2018 as LMG P-30603). The J115strain is the type strain of the species Dysosmobacter welbionis(previously referred to as Dusodibacter welbiota).

In one embodiment, the GC content in the genome of the bacterium of theinvention ranges from about 50 to about 70%, preferably ranges fromabout 55 to 65%, more preferably is of about 59%.

Methods for determining the GC content (i.e. the proportion ofGuanine-Cytosine in a DNA sequence) of a genome are well known to aperson skilled in the art and include, but are not limited to, wholegenome sequencing, High-Pressure Liquid Chromatography, DNA meltingtemperature analysis, and Flow cytometry.

The genome sequence of Dysosmobacter welbionis strain J115, SEQ ID NO:10, has been deposited under the GenBank/EMBL/DDBJ accession numberCP034413.

In one embodiment, the genome sequence of the bacterium of the inventionhas the sequence SEQ ID NO: 10, or has a sequence presenting at leastabout 65% identity with SEQ ID NO: 10, preferably at least about 70%,75%, 80%, 85%, 90% identity with SEQ ID NO: 10, more preferably at leastabout 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 97.6%, 97.7%,97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%,98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9% or more identity with SEQ ID NO: 10.

In one embodiment, the bacterium of the invention has an AverageNucleotide Identity (ANI) score above about 60, preferably above about74, 75, 80, 85, 90, more preferably above about 95, even more preferablyabove about 96, 97, 98, 98.5, 98.65, 99 or more when compared to thegenome of sequence SEQ ID NO: 10.

Techniques to determine the ANI value are known to the person skilled inthe art (such as, for example, methods implemented in Kim et al., Int JSyst Evol Microbiol. 2014 February; 64(Pt 2):346-51). Briefly, ANIcorrespond to corresponds to the sum for each bidirectional best hit(BBH—orthologs sequences identified on the basis of their position inthe genome and sequence identity) of the identity multiplied by thelength of the alignment divided by the total length of BBH genes.

In one embodiment, the bacterium of the invention of the invention has ahybrid DNA-DNA hybridization value (also referred to as DDH value) withSEQ ID NO: 10 above about 60%, preferably above about 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, more preferably above about 70%.

Techniques to determine a DDH value are known to the person skilled inthe art (such as, for example, methods reviewed by Rossello-Móra, inStackebrandt et al., Molecular Identification, Systematics, andPopulation Structure of Prokaryotes, p 23-50, 2006, Springer, Berlin,Heidelberg) and rely on the following general principle: (i) shearingthe genomic DNA (gDNA) of the assayed organism and the gDNA of thereference organism(s) (for instance in the context of the presentinvention the type strain J115 (deposited at the BCCM/LMG on Mar. 14,2018 as LMG P-30603) into small fragments of 600-800 bp; (ii) heatingthe mixture of DNA fragments from both strains to dissociate the DNAdouble-strands; and (iii) subsequently decreasing the temperature untilthe fragments reanneal. For the reason that the melting temperature of adouble-strand depends on the degree of matching base pairings betweenboth strands, genomic (dis-) similarity can be inferred from the meltingtemperature. The hybrid DDH value is usually specified relative to theDDH value obtained by hybridizing a reference genome with itself. DDHvalues ≤70% may be considered as an indication that the tested organismbelongs to a different species than the type strain used as reference.The DDH value may also evaluated on the basis of the genomic sequence ofthe strains to be compared using in publicly available computer programssuch as for example using method described in Meier-Kolthoff et al. (BMCBioinformatics. 2013 Feb. 21; 14:60).

In one embodiment, the bacterium of the invention has an intergenomedistance with SEQ ID NO: 10, below about 0.5, preferably below about0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14 more preferablybellow about 0.13, 0.12, 0.11, 0.10, or less.

Techniques to determine the intergenome distance, or genome-genomedistance (GGD), are known to the skilled artisan. For example, methodsdescribed by Meier-Kolthoff et al. (BMC Bioinformatics 2013; 21:14-60;Int J Syst Evol Microbiol 2014; 1:352-6) may be used. Such method may beimplemented using the genome calculator 2.1 (Deutsche Sammlung vonMikroorganismen and Zellkulturen—DSMZ) using BLAST+ as a local alignmenttool and the sum of all identities found in high-scoring segment pairs(HSP) divided by overall HSP length.

The invention also relates to variants of the bacterium of the inventiondescribed hereinabove. Said variant may also be referred as a derivedstrain of the bacterium of the invention. In one embodiment, the variantof the bacterium of the invention may be obtained by mutation, variationor recombination of the bacterium described herein. In the presentinvention, a variant of a bacterium of the invention may also bereferred to as a mutant of a bacterium of the invention.

In one embodiment, the variant of the bacterium of invention is avariant of Dysosmobacter welbionis.

In one embodiment, the variant of the bacterium of the invention is avariant of strain J115 (deposited at the BCCM/LMG on Mar. 14, 2018 asLMG P-30603).

In one embodiment, a variant of the bacterium of the invention has agenome at least about 70%, preferably at least about 80%, at least about90%, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or more identical to the genome of the bacterium from which itderives.

In one embodiment, the genome sequence of a variant of the bacterium ofthe invention has at least about 65% identity with sequence of thegenome of the bacterium from which it derives, preferably at least about70%, 75%, 80%, 85%, 90% identity with sequence of the genome of thebacterium from which it derives, more preferably at least about 91%,92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%,98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, 98.7%, 98.8%,98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9%, or more, identity with the sequence of the genome of thebacterium from which it derives.

In one embodiment, the genome sequence of a variant of the bacterium ofthe invention has at least about 65% identity with SEQ ID NO: 10,preferably at least about 70%, 75%, 80%, 85%, 90% identity with SEQ IDNO: 10, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%,96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%,98.3%, 98.4%, 98.5%, 98.6%, 98.65%, 98.7%, 98.8%, 98.9%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more,identity with SEQ ID NO: 10.

In one embodiment, a variant of the bacterium of the invention has a 16SrRNA gene sequence having at least about 90% identity with the 16S rRNAgene of the bacterium from which it derives, preferably at least about91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%,97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, 98.7%,98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, 99.9%, or more, identical to the sequence of the 16S rRNA gene ofthe bacterium from which it derives.

In one embodiment, a variant of the bacterium of the invention has a 16SrRNA gene sequence having at least about 99.9% identity with the 16SrRNA gene of the bacterium from which it derives, preferably at leastabout 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%,99.99%, or more, identical to the sequence of the 16S rRNA gene of thebacterium from which it derives.

In one embodiment, a variant of the bacterium of the invention has a 16SrRNA gene sequence having at least about 90% identity with SEQ ID NO: 1,preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 96.5%, 97%,97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%,98.5%, 98.6%, 98.65%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or more, identical to SEQ IDNO: 1.

In one embodiment, a variant of the bacterium of the invention has a 16SrRNA gene sequence having at least about 99.9% identity with SEQ ID NO:1, preferably at least about 99.91%, 99.92%, 99.93%, 99.94%, 99.95%,99.96%, 99.97%, 99.98%, 99.99%,or more, identity with SEQ ID NO: 1.

In one embodiment, a variant of the bacterium of the invention has a 16SrRNA gene sequence having at least about 90% identity with SEQ ID NO: 1over its entire length, preferably at least about 91%, 92%, 93%, 94%,95%, 96%, 96.5%, 97%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%,98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.65%, 98.7%, 98.8%, 98.9%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%,99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, or more,identity with SEQ ID NO: 1 over its entire length.

In one embodiment, a variant of the bacterium of the invention has ahybrid DNA-DNA hybridization value (also referred to as DDH value) aboveabout 60%, preferably above about 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, more preferably above about 70%.

In one embodiment, a variant of the bacterium of the invention has ahybrid DNA-DNA hybridization value (also referred to as DDH value) withthe genome of the bacterium from which it derives above about 60%,preferably above about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, morepreferably above about 70%.

In one embodiment, a variant of the bacterium of the invention has ahybrid DNA-DNA hybridization value (also referred to as DDH value) withSEQ ID NO: 10 above about 60%, preferably above about 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, more preferably above about 70%.

In one embodiment, a variant of the bacterium of the invention has anAverage Nucleotide Identity (ANI) value above about 60%, preferablyabove about 65%, 70%, 75%, 80%, 85%, 90%, more preferably above about95%, even more preferably above about 96%.

In one embodiment, a variant of the bacterium of the invention has anAverage Nucleotide Identity (ANI) score above about 60, preferably aboveabout 74, 75, 80, 85, 90, more preferably above about 95, even morepreferably above about 96, 97, 98, 98.5, 98.65, 99 or more when comparedto the genome sequence of the bacterium from which it derives.

In one embodiment, a variant of a bacterium of the invention has anAverage Nucleotide Identity (ANI) score above about 60, preferably aboveabout 74, 75, 80, 85, 90, more preferably above about 95, even morepreferably above about 96, 97, 98, 98.5, 98.65, 99 or more when comparedto SEQ ID NO: 10.

In one embodiment, a variant of the bacterium of the invention has anintergenome distance with the genome of the bacterium from with derives,below about 0.5, preferably below about 0.22, 0.21, 0.20, 0.19, 0.18,0.17, 0.16, 0.15, 0.14 more preferably bellow about 0.13, 0.12, 0.11,0.10, or less.

In one embodiment, a variant of the bacterium of the invention has anintergenome distance with SEQ ID NO: 10, below about 0.5, preferablybelow about 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14 morepreferably bellow about 0.13, 0.12, 0.11, 0.10, or less.

In one embodiment, the GC content in the genome of a variant of thebacterium of the invention ranges from about 50 to about 70%, preferablyranges from about 55 to 65%, more preferably is of about 59%.

In one embodiment, a variant of the bacterium of the invention is ableto ferment myo-inositol.

In one embodiment, a variant of the bacterium of the invention is unableto ferment D-glucose and/or D-Xylose.

In one embodiment, the diagnostic diamino acid in the cell wall of avariant of the bacterium of the invention is meso-2,6-diaminopimelicacid.

In one embodiment, a variant of the bacterium of the invention has thesame function and/or therapeutic properties as the bacterium from whichit derives.

In one embodiment, the bacterium of the invention and/or a variantthereof is a viable cell. In another embodiment, the bacterium of theinvention and/or a variant thereof is a non-viable cell.

As used herein, the term “viable cells” refers to cells that are able toproliferate in opposition to non-viable cells that are not able toproliferate. Methods for measuring cell viability and proliferation areknown to one skilled in the art. For example, cell viability andproliferation may be assessed by spreading a solution containing atleast one bacterium of the invention across a petri dish and countingthe number of colonies after a determined time of incubation in optimalgrowth conditions. Alternatively, cells may be grown in liquid medium,and proliferation may be measured by measuring optical density of thebacterial culture after a determined time of incubation in optimalgrowth conditions. It is also possible to determine the number of cells,including viable as well as non-viable cells by microscopic observation.While phase-contrast microscopy is a well-known method to do so, themicrobial cells can be further visualized by specific staining withdyes, fluorescent probes or antibodies to facilitate microscopicobservations or count cells by flow cytometry.

In one embodiment, the bacterium of the invention and/or a variantthereof is able to proliferate. In one embodiment, the bacterium of theinvention and/or a variant thereof is alive. In one embodiment, thebacterium of the invention and/or a variant thereof is metabolicallyactive.

In one embodiment, the bacterium of the invention and/or a variantthereof is fresh. The term “fresh” as used herein mean that thebacterium of the invention was not frozen between its last amplificationphase and its use.

In one embodiment, the bacterium of the invention and/or a variantthereof is pasteurized. In one embodiment, the bacterium of theinvention and/or a variant thereof is a pasteurized bacterium.

In one embodiment, the pasteurized bacterium of the invention and/or avariant thereof was heated at a temperature ranging from about 50° C. toabout 100° C., preferably from about 60° C. to about 95° C., morepreferably from about 70° C. to about 90° C. In one embodiment, thepasteurized bacterium of the invention and/or a variant thereof washeated at a temperature of about 50, 51, 52, 53, 54, 55, 56, 57, 58 or59° C. In another embodiment, the pasteurized bacterium of the inventionand/or a variant thereof was heated at a temperature of about 60, 61,62, 63, 64, 65, 66, 67, 68 or 69° C. In yet another embodiment, thepasteurized bacterium of the invention and/or a variant thereof washeated at a temperature of about 70, 71, 72, 73, 74, 75, 76, 77, 78 or79° C. In yet another embodiment, the pasteurized bacterium of theinvention and/or a variant thereof was heated at a temperature of about80, 81, 82, 83, 84, 85, 86, 87, 88 or 89° C. In yet another embodiment,the pasteurized bacterium of the invention and/or a variant thereof washeated at a temperature of about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99°C. or 100° C.

In one embodiment, the pasteurized bacterium of the invention and/or avariant thereof was not heated at a temperature superior to about 100°C. In a particular embodiment, the pasteurized bacterium of theinvention and/or a variant thereof was not heated at an ultra-hightemperature, such as for example at a temperature ranging from about110° C. to about 140° C. In one embodiment, the pasteurized bacterium ofthe invention and/or a variant thereof was not heated at a temperaturesuperior to about 90° C. Accordingly, in one embodiment of theinvention, the bacterium of the invention and/or a variant thereof wasnot sterilized. Sterilization is a treatment intended to destroy, killor inactivate all life forms and other biological agents. This includesmicroorganisms and their spores as well as viruses and prions. Unlikesterilization, pasteurization is not intended to kill all microorganismsbut is usually applied to food with the aim to reduce the number ofviable pathogens.

In one embodiment of the invention, the pasteurized bacterium of theinvention and/or a variant thereof was heated for at least about 10minutes. In another embodiment of the invention, the pasteurizedbacterium of the invention and/or a variant thereof was heated for atleast about 15, 20, 25, 30, 35 or 45 minutes. In one embodiment, thepasteurized bacterium of the invention and/or a variant thereof washeated for a period from about 10 to about 45 minutes.

In one embodiment, the pasteurized bacterium of the invention and/or avariant thereof was not heated for a short time. In a particularembodiment, the pasteurized bacterium of the invention and/or a variantthereof was not heated for less than about 30 seconds, less than about60 seconds, less than about 90 seconds or less than about 120 seconds.In a preferred embodiment, the pasteurized bacterium of the inventionand/or a variant thereof was not heated for a time of less than about 1minute, preferably for a time of less than about 5, 6, 7, 8, or 9minutes.

In one embodiment, the pasteurized bacterium of the invention and/or avariant thereof was heated at a temperature ranging from about 50° C. toabout 100° C. for at least about 10 minutes. In a particular embodiment,the pasteurized bacterium of the invention and/or a variant thereof washeated to about 60° C. for about 20 or about 30 minutes. In anotherparticular embodiment, the pasteurized bacterium of the invention and/ora variant thereof was heated to about 70° C. for about 20 or about 30minutes. In another particular embodiment, the pasteurized bacterium ofthe invention and/or a variant thereof was heated to about 80° C. forabout 20 or about 30 minutes. In another particular embodiment, thepasteurized bacterium of the invention and/or a variant thereof washeated to about 90° C. for about 20 or about 30 minutes.

In a particular embodiment, the pasteurized bacterium of the inventionand/or a variant thereof was not heated at a temperature superior toabout 110° C. for about 1 to about 120 seconds. In another particularembodiment, the pasteurized bacterium of the invention and/or a variantthereof was not heated at a temperature superior to about 100° C. forabout 1 to about 120 seconds. In another particular embodiment, thepasteurized bacterium of the invention and/or a variant thereof was notheated at a temperature superior to about 90° C. for about 1 to about120 seconds.

In one embodiment, the bacterium of the invention and/or a variantthereof is treated with an ultra high temperature (UHT) treatment.

As used herein, a “UHT” treatment refers to an Ultra-high temperatureprocessing or an ultra-heat treatment (both abbreviated UHT) involvingthe at least partial sterilization of a composition by heating it for ashort time, such as, for example, from about 1 to about 60 seconds,preferably from about 1 to about 30 seconds, more preferably from about1 to about 10 seconds, at a temperature of at least about 135° C.

There are two main types of UHT systems: the direct and indirectsystems. In the direct system, products are treated by steam injectionor steam infusion, whereas in the indirect system, products are heattreated using plate heat exchanger, tubular heat exchanger or scrapedsurface heat exchanger. Combinations of UHT systems may be applied atany step or at multiple steps in the process of product preparation.

In one embodiment, the bacterium of the invention and/or a variantthereof is flash pasteurized. Accordingly, in one embodiment, thebacterium of the invention and/or a variant thereof is treated at atemperature ranging from about 71.5° C. to about 74° C. for a period oftime ranging from about 15 to about 30 seconds.

In another embodiment, the bacterium of the invention and/or a variantthereof is a not fresh. In one embodiment, the bacterium of theinvention and/or a variant thereof is frozen.

As used herein the term ‘frozen’, refers to a bacterium that is cooleddown at or below a temperature allowing a phase transition from liquidto solid in said bacterium. In one embodiment said temperature is about−5°, −20° C., −70° C., −80° C. or −190° C.

In one embodiment, cells recovered from the frozen bacterium of theinvention and/or a variant thereof are viable. In other words, in oneembodiment, the bacterium of the invention and/or a variant thereof isfrozen and viable.

In one embodiment, at least about 50% of the cells recovered from thefrozen bacterium of the invention are viable, preferably at least 60%,65%, 70%, 75%, 80%, 85% of the cells recovered from the frozen bacteriumof the invention are viable, more preferably at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94% or 95% or more of the cells recovered fromthe frozen bacterium of the invention are viable.

Methods to prepare frozen stocks of bacteria from which viable cells canbe recovered are known to the person of the art. Briefly and withoutlimitation, bacteria may grow in a suitable liquid culture medium toreach the desired cell density. The desired volume of bacteriapreparation may be diluted with a sterile glycerol solution for a finalglycerol concentration comprised between 15% v/v to 50% v/v glycerol andtransferred to a container able to sustain cold temperatures such as acryogenic vial. The container may be then cooled down to temperature ator below −70° C.

In one embodiment, cells recovered from the frozen bacterium of theinvention and/or a variant thereof are non-viable. In other words, inone embodiment, the bacterium of the invention and/or a variant thereofis frozen and non-viable.

In one embodiment, less than about 50% of the cells recovered from thefrozen bacterium of the invention are viable, preferably less than about40%, 35%, 30%, 25%, 20%, 15%, of the cells recovered from the frozenbacterium of the invention are viable, more preferably less than about14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5% or less of the cellsrecovered from the frozen bacterium of the invention are viable. In oneembodiment, about 7% of the cells recovered from the frozen bacterium ofthe invention are viable.

In one embodiment, the bacterium of the invention and/or a variantthereof is a not able to proliferate. In one embodiment, the bacteriumof the invention and/or a variant thereof is dead. In one embodiment,the bacterium of the invention and/or a variant thereof is a notmetabolically active or is metabolically inactive.

In one embodiment the bacterium of the invention is heat-inactivated. Inone embodiment, the bacterium of the invention is heat-killed.

In a particular embodiment wherein the bacteria of the invention and/ora variant thereof is dead or non-viable, said bacterium and/or variantthereof was killed by heating. In one embodiment, the heat-inactivatedor heat-killed bacterium of the invention and/or a variant thereof washeated at a temperature of at least about 90° C., preferably at leastabout 100° C., 105° C., 110° C., 115° C. or 120° C., more preferably atleast about 121° C., 125° C., 130° C., 135° C., 140° C. or more. In oneembodiment, the heat-inactivated or heat-killed bacterium of theinvention and/or a variant thereof was heated at a temperature of about90° C., preferably of about 100° C., 105° C., 110° C., 115° C. or 120°C., more preferably of about 121° C., 125° C., 130° C., 135° C. or 140°C.

In one embodiment, the heat-inactivated or heat-killed bacterium of theinvention and/or a variant thereof was heated using a saturating steampressure of at least about 10 psig, preferably at least about 11, 12,13, 14, 15 or more psig. In one embodiment, the dead or non-viablebacterium of the invention and/or a variant thereof was heated using asaturating steam pressure of about 10 psig, preferably of about 11, 12,13, 14 or 15 psig.

In one embodiment, the heat-inactivated or heat-killed bacterium of theinvention and/or a variant thereof was heated for at least about 5minutes, preferably for at least about 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes or more. In one embodiment, theheat-inactivated or heat-killed bacterium of the invention and/or avariant thereof was heated for about 5 minutes, preferably for about 10minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes.

The present invention further relates to fragments of the bacterium ofthe invention and/or a variant thereof.

As used herein, the term “fragment” refers to cellular components,metabolites, secreted molecules or vesicles and compounds resulting fromthe metabolism of the bacterium of the invention and/or a variantthereof and the like. Examples of cellular components include, but arenot limited to, bacterial cell wall components such as peptidoglycan,bacterial nucleic acids such as DNA and RNA, bacterial membranecomponents, and bacterial structural components such as proteins,carbohydrates, lipids and combinations of these such as lipoproteins,glycolipids and glycoproteins, bacterial metabolites, organic acids,inorganic acids, bases, peptides, enzymes and co-enzymes, amino acids,carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids,vitamins, bioactive compounds and metabolites containing an inorganiccomponent. Fragments may be obtained by recovering the supernatant of aculture of the bacterium of the invention or by extracting cellcomponents or cell fractions, metabolites or secreted compounds from aculture of the bacterium of the invention and/or a variant thereof, adegradation product, a component in the isolated form, any mixture ofone or more components derived from the bacterium of the inventionand/or a variant thereof, or one or more components present in thebacterium of the invention and/or a variant thereof that are produced inanother way, such as, for example, using recombinant DNA technology, ina microbial host or in any other (bio)synthetic process.

The present invention further relates to a bacterial populationcomprising bacteria belonging to the genus Dysosmobacter, or to thespecies Dysosmobacter welbionis, as described herein. The presentinvention thus relates to a bacterial population comprising at least onebacterium of the invention and/or a variant thereof.

In one embodiment, the bacterial population of the invention issubstantially pure, i.e., at least about 50% of the bacterial cells ofthe bacterial population are bacterial cells of the invention and/orvariants thereof, preferably at least about 60%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of thebacterial cells of the bacterial population are bacterial cells of theinvention and/or variants thereof.

In one embodiment, the bacterium of the invention and/or a variantthereof is isolated.

As used herein, the term isolated refers to the separation of abacterial cell from a natural, mixed population of living microbes, aspresent in the environment, for example in gut microbiota, water, soil,or skin. Isolated bacteria can be amplified on defined laboratorymedium.

The present invention further relates to a method for isolating abacterium of the invention and/or a variant thereof as described herein.

A non-limiting example of a method for isolating the bacteria of theinvention and/or a variant thereof is provided in the experimental part.

In one embodiment, a feces sample is obtained from a subject andtransferred into an anaerobic chamber (Coy) (containing, for example,100% N2 or 80% N2, 15% CO₂, 5% H₂ as gas atmosphere) and immediatelydiluted (e.g., at a dilution 1/10) in an adapted medium. A non-limitingexample of adapted medium is modified YCFA (Yeast extract—caseinhydrolysate—fatty acids), that may optionally be enriched inantioxidants.

Fecal suspension may then be transferred in tubes hermetically sealed,such as, for example, under an atmosphere of about 20% CO₂—about 80% N₂,or 100% CO₂.

Then, single-cell cultivation may be performed using extinction dilutiontechnique, such that a single vial received on average one cell.

In one embodiment, positive cultures after a period of time ranging fromabout 24 h to about 7 days are spread onto solid plates containing anadapted cell culture medium (such as, for example, the modified YCFAmedium as described herein) and incubated for a period of time rangingfrom about 72 h to about 7 days in anaerobic jars with an O₂-absorbingand CO₂-generating agent. Single colonies may then be picked andtransferred to fresh medium and the process is repeated until thecultures are deemed pure. The purify of the culture may be evaluatedusing methods well-known by the skilled artisan, such as, for example,observation of plated bacteria, microscopic observation, that may becombined with Fluorescent in situ hybridization (FISH) cytometry, PCRand/or by sequencing the 16s rRNA gene, in particular using multiplexednext-generation sequencing (NGS) techniques that allows theidentification and the determination of the relative proportion ofdifferent bacterial species in a sample.

In one embodiment, the bacterium of the invention and/or a variantthereof is detectable by a nucleic acid amplification reaction usingspecifics primers.

In one embodiment, the bacterium of the invention and/or a variantthereof is detectable by a nucleic acid amplification reaction using atleast one primer selected from the group comprising, or consisting of,SEQ ID NO: 17 and SEQ ID NO: 18.

In one embodiment, the bacterium of the invention and/or a variantthereof is detectable by a nucleic acid amplification reaction usingprimers of sequence SEQ ID NO: 17 and SEQ ID NO: 18.

The present invention further relates to a method for cultivating anisolated bacterium as described herein. In one embodiment, said methodcomprises culturing the bacterial cells in an adapted medium in ananaerobic atmosphere (e.g., in anaerobic jars with an O₂-absorbing andCO₂-generating agent) at a temperature of about 37° C.

A non-limiting example of composition of a defined laboratory mediumthat can be used to grow the bacteria of the invention is provided intables 1 and 2 hereinafter.

In one embodiment, the culture of the bacteria of the invention isperformed as described in the example hereinafter. Briefly, and withoutlimitation, the bacteria of the invention may be cultured in suspensionunder a 20% CO₂ —80% N₂ or 100% CO₂ atmosphere, in modified YCFA (Yeastextract—casein hydrolysate—fatty acids) medium as defined in tables 1and 2, at 37° C. for 48 hours.

In one embodiment, the modified YCFA medium comprises soy peptone in anamount ranging from more than 0 g/L to about 20 g/L, preferably fromabout 2 g/L to about 6 g/L, more preferably in an amount of about 4 g/L.

In one embodiment, the modified YCFA medium comprises wheat peptone inan amount ranging from more than 0 g/L to about 20 g/L, preferably fromabout 2 g/L to about 6 g/L, more preferably in an amount of about 4 g/L.

In one embodiment, the modified YCFA medium comprises Na₂CO₃ in anamount ranging from more than 0 g/L to about 20 g/L, preferably fromabout 2 g/L to about 6 g/L, more preferably in an amount of about 4 g/L.

In one embodiment, the modified YCFA medium comprises MgCl₂ in an amountranging from more than 0 mg/L to about 500 mg/L, preferably from about25 mg/L to about 75 mg/L, more preferably in an amount of about 50 mg/L.

In one embodiment, the modified YCFA medium comprises glutathione in anamount ranging from more than 0 g/L to about 5 g/L, preferably fromabout 0.5 g/L to about 1.5 g/L, more preferably in an amount of about 1g/L.

In one embodiment, the modified YCFA medium comprises ascorbate in anamount ranging from more than 0 g/L to about 1 g/L, preferably fromabout 0.25 g/L to about 0.75 g/L, more preferably in an amount of about0.5 g/L.

In one embodiment, the modified YCFA medium comprises uric acid in anamount ranging from more than 0 g/L to about 1.2 g/L, preferably fromabout 0.2 g/L to about 0.4 g/L, more preferably in an amount of about0.3 g/L.

In one embodiment, the modified YCFA medium comprises soy peptone in anamount of about 4 g/L, wheat peptone in an amount of about 4 g/L, Na₂CO₃in an amount of about 50 mg/L, glutathione in an amount of about 1 g/L,ascorbate in an amount of about 1 g/L, ascorbate in an amount of about0.5 g/L and uric acid in an amount of about 0.3 g/L.

In one embodiment, the modified YCFA medium is as defined in Tables 1and 2.

The present invention also relates to a composition comprising,consisting of, or consisting essentially of, at least one bacterium ofthe invention and/or a variant thereof and/or a fragment thereof.

As used herein, the term “consisting essentially of”, with reference toa composition, means that the at least one bacterium of the invention orfragment thereof is the only one agent with a biologic activity withinsaid composition.

In one embodiment, at least about 0.5% of bacterial cells in thecomposition of the invention are cells of the bacterium of the inventionand/or a variant thereof and/or a fragment thereof, preferably at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, of bacterial cells inthe composition of the invention are cells of bacterium of the inventionand/or a variant thereof and/or a fragment thereof.

In one embodiment of the invention, the composition of the inventioncomprises an amount of the bacterium of the invention and/or a variantthereof ranging from about 1·10² to about 1·10¹⁵ cfu/g of thecomposition, preferably from about 1·10⁴ to about 1·10¹² cfu/g of thecomposition, more preferably from about 1·10⁵ to about 1·10¹⁰ cfu/g ofthe composition and even more preferably from about 1·10⁶ to about 5·10⁹cfu/g of the composition. In one embodiment, the composition of theinvention comprises an amount of the bacterium of the invention and/or avariant thereof ranging from about 1·10⁴ to about 1·10¹⁴ cfu/g of thecomposition, preferably from about 1·10⁵ to about 1·10¹³ cfu/g of thecomposition, more preferably from about 1·10⁶ to about 1·10¹² cfu/g ofthe composition, even more preferably from about 1·10⁷ to about 1·10¹¹cfu/g of the composition, from about 1·10⁸ to about 1·10¹⁰ cfu/g of thecomposition, and even more preferably from about 2·10⁸ to about 6·10⁹cfu/g of the composition.

As used herein, “cfu” stands for “colony forming unit”.

In one embodiment of the invention, the composition of the inventioncomprises an amount of the bacterium of the invention and/or a variantthereof ranging from about 1·10² to about 1·10¹⁵ cfu/mL of thecomposition, preferably from about 1·10⁴ to about 1·10¹² cfu/mL of thecomposition, more preferably from about 1·10⁵ to about 1·10¹⁰ cfu/mL ofthe composition and even more preferably from about 1·10⁶ to about 5·10⁹cfu/mL of the composition. In one embodiment, the composition of theinvention comprises an amount of the bacterium of the invention and/or avariant thereof ranging from about 1·10⁴ to about 1·10¹⁴ cfu/mL of thecomposition, preferably from about 1·10⁵ to about 1·10¹³ cfu/mL of thecomposition, more preferably from about 1·10⁶ to about 1·10¹² cfu/mL ofthe composition, even more preferably from about 1·10⁷ to about 1·10¹¹cfu/mL of the composition, 1·10⁸ to about 1·10¹⁰ cfu/mL of thecomposition, and even more preferably from about 2·10⁸ to about 6·10⁹cfu/mL of the composition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of the bacterium of the invention ranging from about1·10⁶ to about 1·10¹⁰ cfu/g or cfu/mL of the composition, preferablyfrom about 1·10⁸ to about 1·10¹⁰ cfu/g or cfu/mL, more preferably fromabout 1·10⁹ to about 1·10¹⁰ cfu/g or cfu/mL. In one embodiment of theinvention, the composition of the invention comprises an amount of thebacterium of the invention ranging from about 1·10⁶ to about 1·10¹¹cfu/g or cfu/mL of the composition, preferably from about 1·10⁸ to about1·10¹¹ cfu/g or cfu/mL, more preferably from about 1·10¹⁰ to about1·10¹¹ cfu/g or cfu/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of the bacterium of the invention and/or a variantthereof ranging from about 1·10² to about 1·10¹⁵ cells/g of thecomposition, preferably from about 1·10⁴ to about 1·10¹² cells/g of thecomposition, more preferably from about 1·10⁵ to about 1·10¹⁰ cells/g ofthe composition and even more preferably from about 1·10⁶ to about 1·10⁹cells/g of the composition. In one embodiment, the composition of theinvention comprises an amount of the bacterium of the invention and/or avariant thereof ranging from about 1·10⁴ to about 1·10¹⁴ cells/g of thecomposition, preferably from about 1·10⁵ to about 1·10¹³ cells/g of thecomposition, more preferably from about 1·10⁶ to about 1·10¹² cells/g ofthe composition, even more preferably from about 1·10⁷ to about 1·10¹¹cells/g of the composition, from about 1·10⁸ to about 1·10¹⁰ cells/g ofthe composition, and even more preferably from about 1·10⁹ to about1·10¹⁰ cells/g of the composition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of the bacterium of the invention and/or a variantthereof ranging from about 1·10² to about 1·10¹⁵ cells/mL of thecomposition, preferably from about 1·10⁴ to about 1·10¹² cells/mL of thecomposition, more preferably from about 1·10⁵ to about 1·10¹⁰ cells/mLof the composition and even more preferably from about 1·10⁶ to about1·10⁹ cells/mL of the composition. In one embodiment, the composition ofthe invention comprises an amount of the bacterium of the inventionand/or a variant thereof ranging from about 1·10⁴ to about 1·10¹⁴cells/mL of the composition, preferably from about 1·10⁵ to about 1·10¹³cells/mL of the composition, more preferably from about 1·10⁶ to about1·10¹² cells/mL of the composition, even more preferably from about1·10⁷ to about 1·10¹¹ cells/mL of the composition, from about 1·10⁸ toabout 1·10¹⁰ cells/mL of the composition, and even more preferably fromabout 1·10⁹ to about 1·10¹⁰ cells/mL of the composition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of the bacterium of the invention ranging from about1·10⁶ to about 1·10¹⁰ cells/g or cells/mL of the composition, preferablyfrom about 1·10⁸ to about 1·10¹⁰ cells/g or cells/mL, more preferablyfrom about 1·10⁹ to about 1·10¹⁰ cells/g or cells/mL. In one embodimentof the invention, the composition of the invention comprises an amountof the bacterium of the invention ranging from about 1·10⁶ to about1·10¹¹ cells/g or cells/mL of the composition, preferably from about1·10⁸ to about 1·10¹¹ cells/g or cells/mL, more preferably from about1·10¹⁰ to about 1·10¹¹ cells/g or cells/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the invention and/ora variant thereof corresponding to an amount of bacterium of theinvention and/or a variant thereof ranging from about 1·10² to about1·10¹⁵ cfu/g of the composition, preferably from about 1·10⁴ to about1·10¹² cfu/g of the composition, more preferably from about 1·10⁵ toabout 1·10¹⁰ cfu/g of the composition and even more preferably fromabout 1·10⁶ to about 1·10⁹ cfu/g of the composition. In one embodiment,the composition of the invention comprises an amount of fragment of thebacterium of the invention and/or a variant thereof corresponding to anamount of bacterium of the invention and/or a variant thereof rangingfrom about 1·10⁴ to about 1·10¹⁴ cfu/g of the composition, preferablyfrom about 1·10⁵ to about 1·10¹³ cfu/g of the composition, morepreferably from about 1·10⁶ to about 1·10¹² cfu/g of the composition,even more preferably from about 1·10⁷ to about 1·10¹¹ cfu/g of thecomposition, from about 1·10⁸ to about 1·10¹⁰ cfu/g of the composition,and even more preferably from about 2·10⁸ to about 6·10⁹ cfu/g of thecomposition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the invention and/ora variant thereof corresponding to an amount of bacterium of theinvention and/or a variant thereof ranging from about 1·10² to about1·10¹⁵ cfu/mL of the composition, preferably from about 1·10⁴ to about1·10¹² cfu/mL of the composition, more preferably from about 1·10⁵ toabout 1·10¹⁰ cfu/mL of the composition and even more preferably fromabout 1·10⁶ to about 1·10⁹ cfu/mL of the composition. In one embodimentof the invention, the composition of the invention comprises an amountof fragment of the bacterium of the invention and/or a variant thereofcorresponding to an amount of bacterium of the invention and/or avariant thereof ranging from about 1·10⁴ to about 1·10¹⁴ cfu/mL of thecomposition, preferably from about 1·10⁵ to about 1·10¹³ cfu/mL of thecomposition, more preferably from about 1·10⁶ to about 1·10¹² cfu/mL ofthe composition, even more preferably from about 1·10⁷ to about 1·10¹¹cfu/mL of the composition. In one embodiment of the invention, thecomposition of the invention comprises an amount of fragment of thebacterium of the invention and/or a variant thereof corresponding to anamount of bacterium of the invention and/or a variant thereof rangingfrom about 1·10⁸ to about 1·10¹⁰ cfu/mL of the composition, preferablyfrom about 2·10⁸ to about 6·10⁹ cfu/mL of the composition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the invention and/ora variant thereof corresponding to an amount of bacterium of theinvention and/or a variant thereof ranging from about 1·10⁶ to about1·10¹⁰ cfu/g or cfu/mL of the composition, preferably from about 1·10⁸to about 1·10¹⁰ cfu/g or cfu/mL, more preferably from about 1·10⁹ toabout 1·10¹⁰ cfu/g or cfu/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the inventioncorresponding to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹¹ cfu/g or cfu/mL of the composition,preferably from about 1·10⁸ to about 1·10¹¹ cfu/g or cfu/mL, morepreferably from about 1·10¹⁰ to about 1·10¹¹ cfu/g or cfu/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the invention and/ora variant thereof corresponding to an amount of bacterium of theinvention and/or a variant thereof ranging from about 1·10² to about1·10¹⁵ cells/g of the composition, preferably from about 1·10⁴ to about1·10¹² cells/g of the composition, more preferably from about 1·10⁵ toabout 1·10¹⁰ cells/g of the composition and even more preferably fromabout 1·10⁶ to about 1·10⁹ cells/g of the composition. In one embodimentof the invention, the composition of the invention comprises an amountof fragment of the bacterium of the invention and/or a variant thereofcorresponding to an amount of bacterium of the invention and/or avariant thereof ranging from about 1·10⁴ to about 1·10¹⁴ cells/g of thecomposition, preferably from about 1·10⁵ to about 1·10¹³ cells/g of thecomposition, more preferably from about 1·10⁶ to about 1·10¹² cells/g ofthe composition, even more preferably from about 1·10⁷ to about 1·10¹¹cells/g of the composition. In one embodiment of the invention, thecomposition of the invention comprises an amount of fragment of thebacterium of the invention and/or a variant thereof corresponding to anamount of bacterium of the invention and/or a variant thereof rangingfrom about 1·10⁸ to about 1·10¹⁰ cells/g of the composition, preferablyfrom about 1·10⁹ to about 1·10¹⁰ cells/g of the composition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the invention and/ora variant thereof corresponding to an amount of bacterium of theinvention and/or a variant thereof ranging from about 1·10² to about1·10¹⁵ cells/mL of the composition, preferably from about 1·10⁴ to about1·10¹² cells/mL of the composition, more preferably from about 1·10⁵ toabout 1·10¹⁰ cells/mL of the composition and even more preferably fromabout 1·10⁶ to about 1·10⁹ cells/mL of the composition. In oneembodiment of the invention, the composition of the invention comprisesan amount of fragment of the bacterium of the invention and/or a variantthereof corresponding to an amount of bacterium of the invention and/ora variant thereof ranging from about 1·10⁴ to about 1·10¹⁴ cells/mL ofthe composition, preferably from about 1·10⁵ to about 1·10¹³ cells/mL ofthe composition, more preferably from about 1·10⁶ to about 1·10¹²cells/mL of the composition, even more preferably from about 1·10⁷ toabout 1·10¹¹ cells/mL of the composition. In one embodiment of theinvention, the composition of the invention comprises an amount offragment of the bacterium of the invention and/or a variant thereofcorresponding to an amount of bacterium of the invention and/or avariant thereof ranging from about 1·10⁸ to about 1·10¹⁰ cells/mL of thecomposition, preferably from about 1·10⁹ to about 1·10¹⁰ cells/mL of thecomposition.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the inventioncorresponding to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹⁰ cells/g or cells/mL of the composition,preferably from about 1·10⁸ to about 1·10¹⁰ cells/g or cells/mL, morepreferably from about 1·10⁹ to about 1·10¹⁰ cells/g or cells/mL.

In one embodiment of the invention, the composition of the inventioncomprises an amount of fragment of the bacterium of the inventioncorresponding to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹¹ cells/g or cells/mL of the composition,preferably from about 1·10⁸ to about 1·10¹¹ cells/g or cells/mL, morepreferably from about 1·10¹⁰ to about 1·10¹¹ cells/g or cells/mL.

In one embodiment, the bacterium of the invention and/or a variantthereof is pasteurized, and the amounts recited herein corresponds toamounts before the step of pasteurization.

In one embodiment, the bacterium of the invention and/or a variantthereof is frozen, and the amounts recited herein corresponds to amountsbefore the freezing step. In another embodiment, the bacterium of theinvention and/or a variant thereof is frozen, and the amounts recitedherein corresponds to amounts after the freezing step.

The present invention also relates to at least one bacterium and/orvariant thereof as described herein above as a probiotic. In oneembodiment, the at least one bacterium of the invention, preferably thestrain J115, and/or variant thereof is probiotics, which is beneficialfor improving the gastrointestinal environment of a subject.

The present invention further relates to the therapeutic use of at leastone bacterium of the invention and/or a variant thereof and/or afragment thereof.

In one embodiment, the composition of the invention is a pharmaceuticalcomposition or a medicament.

The present invention thus also relates to a pharmaceutical compositioncomprising, consisting essentially of or consisting of the bacterium orthe composition according to the invention and at least onepharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients that may be used in thecompositions of the invention include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, trehalose, buffer substances such as phosphates,glycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes, such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, silica, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances(for example sodium carboxymethylcellulose), polyethylene glycol,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

In one embodiment, the pharmaceutical composition is for treating and/orpreventing a disease related to the gut microbiota, preferably ametabolic disease.

The present invention further relates to a medicament comprising,consisting essentially of or consisting of the bacterium or thecomposition according to the invention. The invention also relates tothe use of a bacterium or composition according to the invention for themanufacture of a medicament.

In one embodiment, the medicament is for treating and/or preventing adisease related to the gut microbiota, preferably a metabolic disease.

In one embodiment, the pharmaceutical composition or medicament of theinvention may further contain antioxidant agents such as ascorbic acid,ascorbyl palmitate, BHT, potassium sorbate or Rosmarinus officinalisextracts.

In one embodiment, the pharmaceutical composition or medicament of theinvention may further contain flavour agents such as sugars, fruit ortea flavourings.

In one embodiment, composition comprising at least one bacterium of theinvention and/or fragments thereof can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose.

In one embodiment, composition comprising at least one bacterium of theinvention and/or fragments thereof can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils.

In one embodiment, the composition comprising at least one bacterium ofthe invention and/or fragments thereof may comprise a carrier that canalso be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetables oils such as oleic acid.

The proper fluidity can be maintained, for example, by the use of acoating, such as lecithin (i.e., Soy lecithin or de-greased soylecithin), by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants.

In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above.

Upon formulation, the pharmaceutical composition or the medicament ofthe invention will be administered in a manner compatible with thedosage formulation and in such amount as is effective. Thepharmaceutical composition or the medicament of the invention may beadministered in a variety of dosage forms, such as drug release capsulesand the like. Some variation in dosage will necessarily occur dependingon the condition of the subject being treated. The person responsiblefor administration will, in any event, determine the appropriate dosefor the individual subject.

The pharmaceutical composition and medicament of the present invention,the bacterium of the invention and/or a variant thereof, and/or fragmentthereof, alone or in combination with another active principle, can beadministered in a unit administration form, as a mixture withconventional pharmaceutical supports, to animals and human beings.Suitable unit administration forms comprise oral-route forms such astablets, gel capsules, powders, granules and oral suspensions orsolutions, sublingual and buccal administration forms, aerosols,implants, subcutaneous, transdermal, topical, intraperitoneal,intramuscular, intravenous, subdermal, transdermal, intrathecal andintranasal administration forms and rectal administration forms.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, the pharmaceuticalcomposition or the medicament of the invention is to be administered, oris adapted to be administered, systemically or locally.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition or medicament of the invention is to be administered, or isadapted to be administered, orally, buccally, by injection, bypercutaneous administration, parenterally, intraperitoneal, byendoscopy, topically, transdermally, transmucosally, nasally, byinhalation spray, rectally, vaginally, intratracheally, and via animplanted reservoir, or any combination thereof.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition or medicament of the invention is to be orally administered,or is adapted to be orally administered. Examples of formulationsadapted to oral administration include, but are not limited to, solidforms, liquid forms and gels. Examples of solid forms adapted to oraladministration include, but are not limited to, pill, tablet, capsule,soft gelatine capsule, hard gelatine capsule, dragees, granules, caplet,compressed tablet, cachet, wafer, sugar-coated pill, sugar coatedtablet, or dispersing/or disintegrating tablet, powder, solid formssuitable for solution in, or suspension in, liquid prior to oraladministration and effervescent tablet. Examples of liquid form adaptedto oral administration include, but are not limited to, solutions,suspensions, drinkable solutions, elixirs, sealed phial, potion, drench,syrup, liquor and sprays.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition or medicament of the invention is to be administeredrectally, or is adapted to be rectally administered. Example offormulations adapted to rectal administration include, but are notlimited to: suppository, micro enemas, enemas, gel, rectal foam, cream,ointment.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition or medicament of the invention is to be injected, or isadapted to be injected. Examples of formulations adapted to injectionsinclude, but are not limited to, liquid solutions or suspensions, solidforms suitable for solution in, or suspension in, liquid prior toinjection.

Examples of systemic injections include, but are not limited to,intravenous, intratumoral, intracranial, intralymphatic,intraperitoneal, intramuscular, subcutaneous, intradermal,intraarticular, intrasynovial, intrasternal, intrathecal, intravesical,intrahepatic, intralesional, intracavernous, infusion techniques andperfusion. In another embodiment, when injected, the composition, thepharmaceutical composition or the medicament of the invention issterile.

Methods for obtaining a sterile pharmaceutical composition include, butare not limited to, GMP synthesis (GMP stands for “Good manufacturingpractice”).

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition or medicament of the invention is to be administered, or isadapted to be administered, in an immediate release form. In oneembodiment, the bacterium of the invention and/or a variant thereofand/or fragments thereof, the composition, pharmaceutical composition ormedicament of the invention is to be administered, or is adapted to beadministered, in a mixed-release form. In one embodiment, the bacteriumof the invention and/or a variant thereof and/or fragments thereof, thecomposition, pharmaceutical composition or medicament of the inventionis to be administered, or is adapted to be administered, in anenterically-coated form. In one embodiment, the bacterium of theinvention and/or a variant thereof and/or fragments thereof, thecomposition, pharmaceutical composition or medicament of the inventionis to be administered, or is adapted to be administered, in asustained-release form.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition or medicament of the invention comprises a delivery systemthat controls the release of the active ingredients.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention further comprises at least oneadditional probiotic strain or species, such as, for example, bacterialprobiotic strains or species; prokaryotes probiotics other thanbacteria; or fungal strains or species, preferably yeast strains orspecies. In one embodiment, said additional probiotic strains or speciesare selected from those naturally present in the gut of the subject,preferably in the human gut, more preferably in the gut of substantiallyhealthy human subjects. In one embodiment, said additional probioticstrains or species are selected for strains or species not accordingnaturally in the gut of the subject such as those found for instance indairy products.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention further comprises at least one bacterialprobiotic strain or species selected from the group comprising orconsisting of Lactobacillus, Lactococcus, Akkermansia, Bifidobacterium,Veillonella, Desemzia, Christensenella, Allobaculum, Coprococcus,Collinsella, Citrobacter, Turicibacter, Sutterella, Subdoligranulum,Streptococcus, Sporobacter, Sporacetigenium, Ruminococcus, Roseburia,Proteus, Propionibacterium, Leuconostoc, Weissella, Pediococcus,Streptococcus, Prevotella, Parabacteroides, Papillibacter, Oscillospira,Melissococcus, Dorea, Dialister, Clostridium, Cedecea, Catenibacterium,Butyrivibrio, Buttiauxella, Bulleidia, Bilophila, Bacteroides,Anaerovorax, Anaerostopes, Anaerofilum, Enterobacteriaceae, Fermicutes,Atopobium, Alistipes, Acinetobacter, Slackie, Shigella, Shewanella,Serratia, Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium,Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, andEggerthella or a mixture thereof.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention further comprises at least oneprokaryote strain or species selected from the group comprising orconsisting of Archaea, Firmicutes, Verrucomicrobia (such as, forexample, Akkermansia muciniphila), Christensenella, Bacteroidetes (suchas, for example, Allistipes, Bacteroides ovatus, Bacteroidessplachnicus, Bacteroides stercoris, Bacteroides vulgatus,Parabacteroides, Prevotella ruminicola, Porphyromondaceae, and relatedgenus), Proteobacteria, Betaproteobacteria (such as, for example,Aquabacterium and Burkholderia), Gammaproteobacteria (such as, forexample, Xanthomonadaceae), Actinobacteria (such as, for example,Actinomycetaceae and Atopobium), Methanobacteria, Spirochaetes,Fibrobacteres, Deferribacteres, Deinococcus, Thermus, Cyanobacteria,Methanobrevibacteria, Ruminococcus, Coprococcus, Subdolingranulum,Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Dialister,Anaerotruncus, Staphylococcus, Micrococcus, Propionibacteria,Enterobacteriaceae, Faecalibacterium, Bacteroides, Parabacteroides,Prevotella, Eubacterium, Bacilli (such as, for example, Lactobacillussalivarius and related species, Aerococcus, Granulicatella,Streptococcus bovis and related genus and Streptococcus intermedius andrelated genus), Clostridium (such as, for example, Eubacterium hallii,Eubacterium limosum and related genus) and Butyrivibrio or a mixturethereof.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention further comprises at least one fungalprobiotic strain or species, preferably yeast probiotic strain orspecies, selected from the group comprising or consisting ofAscomycetes, Zygomycetes and Deuteromycetes, preferably from the groupsAspergillus, Torulopsis, Zygosaccharomyces, Hansenula, Candida,Saccharomyces, Clavispora, Bretanomyces, Pichia, Amylomyces,Zygosaccharomyces, Endomyces, Hyphopichia, Zygosaccharomyces,Kluyveromyces, Mucor, Rhizopus, Yarrowia, Endomyces, Debaryomyces, andPenicillium or a mixture thereof.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention further comprises at least oneprebiotic.

In one embodiment, the composition, the pharmaceutical composition orthe medicament of the invention further comprises at least one prebioticselected from the group comprising or consisting of myo-inositol, inulinand inulin-type fructans, oligofructose, beta-glucans, xylose,arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose,fructose, lactose, salicin, sucrose, glucose, esculin, tween 80,trehalose, maltose, mannose, mellibiose, mucus or mucins, raffinose,fructooligosaccharides, galacto-oligosaccharides, amino acids, alcohols,fermentable carbohydrates and any combinations thereof.

In a particular embodiment, the composition, the pharmaceuticalcomposition or the medicament of the invention further comprisesmyo-inositol.

Other non-limiting examples of prebiotics include water-solublecellulose derivatives, water-insoluble cellulose derivatives,unprocessed oatmeal, metamucil, all-bran, polyphenols and anycombinations thereof.

Examples of water-soluble cellulose derivatives include, but are notlimited to, methylcellulose, methyl ethyl cellulose, hydroxyethylcellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethylcellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose,hydroxypropyl methylcellulose, and carboxymethyl cellulose.

The present invention also relates to a therapeutic combination productfor its separate, simultaneous or sequential administration.

As used herein the term “therapeutic combination product” (that may alsobe referred to as a therapeutic kit of parts) refers to a productcomprising or consisting of at least the 2 following parts: a first partcomprising (preferably in a therapeutically affective amount) abacterium of the invention and/or a variant thereof, a compositionaccording to the invention, a pharmaceutical composition according tothe invention, or a medicament according to the invention and a secondpart comprising a composition comprising at least one probiotic and/orat least one prebiotic and/or one other drug for a therapeutic use. Inone embodiment, the therapeutic combination product is for treatingand/or preventing a disease related to the gut microbiota, preferably ametabolic disease.

Examples of other drugs known for their use in treating and/orpreventing metabolic diseases include, but are not limited to,sulphonylurea (such as, for example, acetohexamide, carbutamide,chlorpropamide, glycyclamide, metahexamide, tolazamide, tolbutamide,glibenclamide, glibornuride, gliclazide, glipizide, gliquidone,glisoxepide, glyclopyramide and glimepiride and their derivatives);biguanides (such as, for example, metformin, pheformin and buformin);alpha-glucosidase inhibitors (such as, for example, acarbose, miglitoland voglibose); thiazolidinedione (such as, for example, pioglitazone,rosiglitazone and lobeglitazone); HMG-CoA reductase inhibitors (orstatins) (such as, for example, simvastatin, pravastatin, atorvastatin,mevastatin, cerivastatin, rosuvastatin and fluvastatin); dipeptidylpeptidase 4 inhibitors (or gliptins) (such as, for example, sitagliptin,vildagliptin, sawagliptin, linagliptin, gemigliptin, anagliptin,teneligliptin, alogliptin, trelagliptin, omarigliptin, evogliptin,gosopliptin, dutogliptin and berberine); glucagon-like peptide analogsand agonists (such as, for example, exenatide, liraglutide andlixisenatide); sodium/glucose cotransporter 2 inhibitors (such as, forexample, empagliflozin, dapagliflozin, canagliflozin, ipragliflozin,ertugliflozin, luseogliflozin, bexagliflozin, todogliflozin,henagliflozin, sotagliflozin, remogliflozin, sergliflozin andatigliflozin); cholestyramine, colesevelam, colestipol and ezetimibe.

In one embodiment, the at least two parts of the therapeutic combinationproduct according to the invention are administered simultaneously. Inanother embodiment, the at least two parts of the therapeuticcombination product according to the invention are administered at adifferent time. In another embodiment, at least two parts of thetherapeutic combination product according to the invention areadministered sequentially.

In one embodiment, the at least two parts of the therapeutic combinationproduct according to the invention are administered using differentadministration routes (such as, for example, one part by oraladministration, and the second one by injection). In another embodiment,the at least two parts of the therapeutic combination product accordingto the invention are administered using the same administration route(such as, for example, oral administration or injection).

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, pharmaceuticalcomposition, the therapeutic combination product of the invention is foruse as a medicament.

The present invention further relates to at least one bacterium of theinvention and/or a variant thereof and/or fragments thereof, or to thecomposition, pharmaceutical composition, medicament or therapeuticcombination product of the invention for use in the treatment and/orprevention of disorders related to the gastrointestinal microbiota in asubject in need thereof.

Example of disorders related to the gastrointestinal microbiota include,but are not limited to, metabolic diseases (such as for example,obesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, Diabetes Mellitus (such as, for example, Type 2Diabetes), glucose intolerance, hyperglycemia, abnormal lipidmetabolism, dyslipidemia, high cholesterol, elevated LDL-cholesterol,decreased LDL cholesterol, elevated triglycerides, adipose tissuesinflammation and adipose tissue fibrosis, infections, colitis (such asfor example, inflammatory bowel disease (e.g., Crohn's disease andulcerative colitis), ischemic colitis, irritable bowel syndrome,lymphocytic colitis and collagenous colitis), cancers (such as forexample, colorectal cancer), dysfunction of the immune system (such asfor example, eczema, allergies, food allergies and celiac disease),psychological disorders (such as for example, stress, anxiety andaddiction), neurological disorders (such as for example, Parkinson'sdisease and Alzheimer's disease), liver diseases (such as for example,cirrhosis, non-alcoholic fatty liver disease, and hepatic steatosis),cachexia, Prader-Willy syndrome, dysfunction of the digestive tract(such as for example, ulcers and gallbladder disease), feeding behaviorsdisorders (such as for example, anorexia nervosa, bulimia nervosa andbinge-eating disorder), cardiovascular diseases and conditions (such as,for example strokes, atherosclerosis and hypertension), asthma, sleepapnea, osteoarthritis and inflammatory diseases.

In one embodiment, at least one bacterium of the invention and/or avariant thereof and/or fragments thereof, the composition, thepharmaceutical composition, the medicament or the therapeuticcombination product of the invention is for, or for use in, thetreatment and/or prevention of diseases selected from the groupcomprising, or consisting of, metabolic diseases, obesity, metabolicsyndrome, insulin-deficiency or insulin-resistance related disorders,Diabetes Mellitus, type 2 diabetes, type 1 diabetes, glucoseintolerance, hyperglycemia, abnormal lipid metabolism, dyslipidemia,high cholesterol, elevated LDL-cholesterol, decreased LDL cholesterol,elevated triglycerides, adipose tissues inflammation, adipose tissuefibrosis, infections, colitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, ischemic colitis, irritable bowel syndrome,lymphocytic colitis, collagenous colitis, enteritis, cancers, colorectalcancer, dysfunction of the immune system, eczema, allergies, foodallergies, celiac disease, psychological disorders, stress, anxiety,addiction, neurological disorders, Parkinson's disease, Alzheimer'sdisease, liver diseases, cirrhosis, non-alcoholic fatty liver disease,hepatic steatosis, cachexia, Prader-Willy syndrome, dysfunction of thedigestive tract, ulcers, gallbladder disease, feeding behaviorsdisorders, anorexia nervosa, bulimia nervosa, binge-eating disorder,cardiovascular diseases, strokes, atherosclerosis and hypertension,asthma, sleep apnea, osteoarthritis and inflammatory diseases.

In one embodiment, at least one bacterium of the invention and/or avariant thereof and/or fragments thereof, the composition, thepharmaceutical composition, the medicament or the therapeuticcombination product of the invention is for, or for use in, thetreatment and/or prevention of diseases selected from the groupcomprising, or consisting of, metabolic diseases, obesity, metabolicsyndrome, insulin-deficiency or insulin-resistance related disorders,Diabetes Mellitus, type 2 diabetes, type 1 diabetes, glucoseintolerance, hyperglycemia, abnormal lipid metabolism, dyslipidemia,high cholesterol, elevated LDL-cholesterol, decreased LDL cholesterol,elevated triglycerides, adipose tissues inflammation, adipose tissuefibrosis, infections, colitis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, ischemic colitis, irritable bowel syndrome,lymphocytic colitis, collagenous colitis, enteritis, food allergies,celiac disease, ulcers, cachexia, Prader-Willy syndrome, feedingbehaviors disorders and binge-eating disorder.

In one embodiment, at least one bacterium of the invention and/orvariant thereof and/or fragments thereof, the composition, thepharmaceutical composition, the medicament or the therapeuticcombination product of the invention is for, or for use in, thetreatment and/or prevention of diseases selected from the groupcomprising, or consisting of, metabolic diseases, infections, colitis,enteritis, food allergies, celiac disease, ulcers, cachexia,Prader-Willy syndrome and feeding behaviors disorders.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a metabolic disease.

Example of metabolic diseases include, but are not limited to obesity,metabolic syndrome, insulin-deficiency or insulin-resistance relateddisorders, Diabetes Mellitus (such as, for example, Type 2 Diabetes),glucose intolerance, abnormal lipid metabolism, hyperglycemia,dyslipidemia, high cholesterol, elevated LDL-cholesterol, decreasedHDL-cholesterol, elevated triglycerides, adipose tissues inflammationand adipose tissues fibrosis.

In one embodiment, at least one bacterium of the invention and/or avariant thereof and/or fragments thereof, the composition, thepharmaceutical composition, the medicament or the therapeuticcombination product of the invention is for, or for use in, thetreatment and/or prevention of metabolic diseases selected from thegroup comprising, or consisting of, obesity, metabolic syndrome,insulin-deficiency or insulin-resistance related disorders, DiabetesMellitus, type 2 diabetes, type 1 diabetes, glucose intolerance,hyperglycemia, abnormal lipid metabolism, dyslipidemia, highcholesterol, elevated LDL-cholesterol, decreased LDL cholesterol,elevated triglycerides, adipose tissues inflammation and adipose tissuefibrosis.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a metabolic disease, preferably selected from the groupcomprising, or consisting of, obesity, Diabetes Mellitus, preferablyType 2 Diabetes Mellitus, metabolic syndrome, insulin-deficiency orinsulin-resistance related disorders and glucose intolerance.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is obesity. In one embodiment, the disorder related to thegastrointestinal microbiota is Type 2 Diabetes Mellitus.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a metabolic disease, preferably selected from the groupcomprising, or consisting of, abnormal lipid metabolism, hyperglycemia,dyslipidemia, high cholesterol, elevated LDL-cholesterol, decreasedHDL-cholesterol and elevated triglycerides.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is an infection.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is colitis, preferably selected from the group comprising, orconsisting of, inflammatory bowel disease (e.g., Crohn's disease andulcerative colitis), ischemic colitis, irritable bowel syndrome,lymphocytic colitis and collagenous colitis.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is cancer, preferably colorectal cancer.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a dysfunction of the immune system, preferably selectedfrom the group comprising eczema, allergies, food allergies and celiacdisease.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is selected from the group comprising, or consisting of, foodallergies and celiac disease.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a psychological disorder, preferably selected from thegroup comprising, or consisting of, stress, anxiety, and addiction.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a neurological disorder, preferably selected from thegroup comprising, or consisting of, Parkinson's disease and Alzheimer'sdisease.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a liver disease, preferably selected from the groupcomprising, or consisting of, cirrhosis, non-alcoholic fatty liverdisease, and hepatic steatosis.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is cachexia.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is Prader-Willy syndrome.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a dysfunction of the digestive tract, preferably selectedfrom the group comprising, or consisting of, ulcers and gallbladderdisease.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a feeding behavior disorder preferably selected from thegroup comprising, or consisting of, anorexia nervosa, bulimia nervosaand binge-eating disorder.

In one embodiment, the disorder related to the gastrointestinalmicrobiota is a cardiovascular disease or condition, preferably selectedfrom the group comprising, or consisting of, strokes, atherosclerosisand hypertension.

In one embodiment, at least one bacterium of the invention and/or avariant thereof and/or fragments thereof, the composition, thepharmaceutical composition, the medicament or the therapeuticcombination product of the invention is for, or for use in, thetreatment and/or prevention of inflammatory diseases. In one embodiment,the inflammatory disease is selected form the group comprising, orconsisting of, adipose tissue inflammation, adipose tissue dysfunction,colitis and enteritis.

The inventors have observed that the presence of the bacteria of theinvention in the gut microbiota promote the integrity of the gutepithelial barrier. Beyond its importance in metabolic function, notablyin type 1 diabetes, a decrease in the integrity of the gut epithelialbarrier has been found associated with several other diseases such asinfection, colitis, enteritis, eczema, allergies, food allergies, liverdiseases, inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), celiac disease, and psychological disorders (e.g.,anxiety, stress and addiction).

The present invention thus further relates to the bacterium of theinvention and/or a variant thereof and/or fragments thereof, or to thecomposition, pharmaceutical composition, medicament or therapeuticcombination product of the invention for, or for use in, the treatmentand/or prevention of a disease related to an increased epithelialbarrier permeability. Examples of diseases related to an increasedepithelial barrier permeability include, but are not limited to,infection, colitis, enteritis, type I diabetes, eczema, allergies, foodallergies, liver diseases, inflammatory bowel diseases (e.g., Crohn'sdisease and ulcerative colitis), celiac disease, and psychologicaldisorders (e.g., anxiety, stress and addiction).

In one embodiment, at least one bacterium of the invention and/or avariant thereof and/or fragments thereof, the composition, thepharmaceutical composition, the medicament or the therapeuticcombination product of the invention is for, or for use in, thetreatment of diseases related to the integrity of the intestinalepithelial barrier. In one embodiment, diseases related the integrity ofthe intestinal epithelial barrier are selected form the groupcomprising, or consisting of, infections, colitis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, ischemic colitis,irritable bowel syndrome, lymphocytic colitis, collagenous colitis,enteritis, celiac diseases and food allergies.

The present invention relates to a method for treating and/or preventinga disorder selected from the group consisting of metabolic diseases,obesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, Diabetes Mellitus, type 2 diabetes, type 1 diabetes,glucose intolerance, hyperglycemia, abnormal lipid metabolism,dyslipidemia, high cholesterol, elevated LDL-cholesterol, decreased LDLcholesterol, elevated triglycerides, adipose tissues inflammation,adipose tissue fibrosis, infections, colitis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, ischemic colitis,irritable bowel syndrome, lymphocytic colitis, collagenous colitis,enteritis, cancers, colorectal cancer, dysfunction of the immune system,eczema, allergies, food allergies, celiac disease, psychologicaldisorders, stress, anxiety, addiction, neurological disorders,Parkinson's disease, Alzheimer's disease, liver diseases, cirrhosis,non-alcoholic fatty liver disease, hepatic steatosis, cachexia,Prader-Willy syndrome, dysfunction of the digestive tract, ulcers,gallbladder disease, feeding behaviors disorders, anorexia nervosa,bulimia nervosa, binge-eating disorder, cardiovascular diseases,strokes, atherosclerosis and hypertension, asthma, sleep apnea,osteoarthritis and inflammatory diseases in a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for treating and/or preventinga disorder selected from the group consisting of metabolic diseases,obesity, metabolic syndrome, insulin-deficiency or insulin-resistancerelated disorders, Diabetes Mellitus, type 2 diabetes, type 1 diabetes,glucose intolerance, hyperglycemia, abnormal lipid metabolism,dyslipidemia, high cholesterol, elevated LDL-cholesterol, decreased LDLcholesterol, elevated triglycerides, adipose tissues inflammation,adipose tissue fibrosis, infections, colitis, inflammatory boweldisease, Crohn's disease, ulcerative colitis, ischemic colitis,irritable bowel syndrome, lymphocytic colitis, collagenous colitis,enteritis, food allergies, celiac disease, ulcers, cachexia,Prader-Willy syndrome, feeding behaviors disorders and binge-eatingdisorder in a subject, comprising administering to the subject at leastone bacterium of the invention and/or a variant thereof and/or at leastone fragment thereof.

The present invention relates to a method for treating and/or preventinga disorder selected from the group consisting of metabolic diseases,infections, colitis, enteritis, food allergies, celiac disease, ulcers,cachexia, Prader-Willy syndrome and feeding behaviors disorders in asubject, comprising administering to the subject at least one bacteriumof the invention and/or a variant thereof and/or at least one fragmentthereof.

The present invention relates to a method for treating and/or preventinga metabolic disease in a subject, comprising administering to thesubject at least one bacterium of the invention and/or a variant thereofand/or at least one fragment thereof. In one embodiment, the method isfor treating and/or preventing a disease selected from the groupcomprising, or consisting of, obesity, Diabetes Mellitus, preferablyType 2 Diabetes Mellitus, metabolic syndrome, insulin-deficiency orinsulin-resistance related disorders and glucose intolerance.

In one embodiment, the method is for treating and/or preventing obesityin a subject in need thereof. In one embodiment, the method is fortreating and/or preventing Type 2 Diabetes Mellitus in a subject in needthereof.

The present invention relates to a method for treating and/or preventinga disorder related to the integrity of the intestinal epithelial barrierpreferably selected from the group consisting of infections, colitis,inflammatory bowel disease, Crohn's disease, ulcerative colitis,ischemic colitis, irritable bowel syndrome, lymphocytic colitis,collagenous colitis, enteritis, celiac diseases and food allergies in asubject, comprising administering to the subject at least one bacteriumof the invention and/or a variant thereof and/or at least one fragmentthereof.

In one embodiment, the subject is a human.

In one embodiment, the subject is/was diagnosed with a disorder relatedto the gastrointestinal microbiota.

In one embodiment, the subject is at risk of developing a disorderrelated to the gastrointestinal microbiota. Examples of risk factors mayinclude, without limitation, the fact that the subject is overweight orobese, or a predisposition, such as, for example, a familialpredisposition to such disorder.

In one embodiment, the subject is obese. As used herein, the term“obese” refers herein to a medical condition wherein the subjectpreferably has a BMI above about 30, preferably above about 35, morepreferably above about 40.

The “BMI” or “body mass index” is defined as the subject's body mass inkilograms divided by the square of his height in meters. The formulaeuniversally used in medicine produce a unit of measure of kg/m².

The inventors have estimated that the proportion of the bacteria of theinvention present in the gut microbiota of a substantially healthysubject ranges from about 0.1% to 5%; preferably from about 0.5% to 4%of the total bacteria found in the feces of the subject, more preferablyfrom about 0.8% to 3%. In one embodiment, the proportion of the bacteriaof the invention present in the gut microbiota of a substantiallyhealthy subject is about 2.45% of the total bacteria found in the fecesof the subject.

In one embodiment of the invention, the subject presents a deregulationof the gut microbiota composition. Preferably, the gut microbiota ofsaid subject is depleted in the bacterium of the invention, morepreferably as compared to the gut microbiota of a substantially healthysubject.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, the pharmaceuticalcomposition, the medicament or the therapeutic combination product ofthe invention is to be administered at a dose determined by the skilledartisan and personally adapted to each subject.

In addition, the specific therapeutically effective amount for anyparticular subject will depend upon a variety of factors including thespecific composition employed, the age, body weight, general health, sexand diet of the subject; the time of administration, route ofadministration, the duration of the treatment; drugs used in combinationor coincidental with the composition of the invention; and like factorswell known in the medical, nutraceutical and cosmetic arts.

In one embodiment, a therapeutically effective amount of the bacteriumof the invention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention is to be administeredat least once a day, at least twice a day, at least three times a day.

In one embodiment, a therapeutically effective amount of the bacteriumof the invention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention is to be administeredevery two, three, four, five, six days.

In one embodiment, a therapeutically effective amount of the bacteriumof the invention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention is to be administeredtwice a week, every week, every two weeks, once a month.

In one embodiment, a therapeutically effective of the bacterium of theinvention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention is to be administeredevery month, every two months, every three months, every four months,every five months, every six months, once a year.

In one embodiment, a therapeutically effective of the bacterium of theinvention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention is to be administeredfor a period of time of about one day, two days, three days, four days,five days, six days, a week, two weeks, three weeks, a month, twomonths, three months, six months, a year, or over longer periods suchas, e.g., for several years or for the rest of the life of the subject.

In one embodiment, when a therapeutic combination product of theinvention is to be administered to the subject, each part of saidcombination product may be administered at a different frequency and fora different period of time.

The specific therapeutically effective amount for any particular subjectwill depend upon a variety of factors including the disease beingtreated and the severity of the disease. For example, it is well withinthe skill of the art to start doses of a therapeutic compound at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved;but, at the opposite, it can be equally useful to start with a loadingdose, a manner to reach steady-state plasma concentration more quickly(also referred to as a bolus), and then, optionally, to follow with amaintenance dose calculated to exactly compensate the effect of theelimination process.

It will be understood that the total daily usage of the bacterium of theinvention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention will be decided by theattending physician within the scope of sound medical judgment.

In one embodiment, a therapeutically effective amount of the bacteriumof the invention and/or a variant thereof and/or fragments thereof, orof the composition, the pharmaceutical composition, the medicament orthe therapeutic combination product of the invention is to beadministered until treatment or alleviation of the disorder related tothe gastrointestinal microbiota; or until the desired therapeutic effecthas been achieved.

Examples of desired therapeutic effect relating to the administration ofthe bacteria of the invention include, but are not limited to,restoration of a normal proportion of the bacterium of the invention inthe gut of a subject, preferably defined by the proportion of thebacterium of the invention measured in the gut of a substantiallyhealthy subject, an increase in the proportion of the bacterium of theinvention in the gut of a subject, an increase of the abundance of anyactive compounds of the bacterium of the invention in the gut of asubject, a decrease of glucose intolerance, a decrease of insulinresistance, a decrease of the permeability of the gut intestinalbarrier, an increase of the expression level of genes involved in theestablishment and/or maintenance of the epithelial barrier, such asgenes coding for occludins, claudins, junctional adhesion molecules(JAM), E-cadherin, catenins, nectin, afadin, zonulin and zonulaoccludens (ZO)-1, ZO-2 and ZO-3, an increase in the secretion of mucusby the intestinal epithelium, an increase of the level of proglucagon,glucagon-like peptide 1 (GLP-1) and/or glucagon-like peptide 2 (GLP-2),a reduction of diet-induced fasting hyperglycemia, an inhibition and/oran activation of the growth and/or biological activity of othermicroorganisms of the gut microbiota in a subject, an assistance in thedefense against exogenous pathogenic bacteria, an assistance indigestion and an increase in the production of antibacterial compound,vitamins, SCFA, acetate, acetic acid, propionate, propionic acid,isopropionate, isopropionic acid valerate, valeric acid, isovalerate,isovaleric acid, isobutyrate, isobutyric acid, butyrate and/or butyricacid in the gut of a subject.

In one embodiment, a therapeutically effective amount of the bacteriumof the invention and/or a variant thereof and/or fragments thereof, thecomposition, the pharmaceutical composition, the medicament or thetherapeutic combination product of the invention is to be administeredfor a chronic treatment. In another embodiment, a therapeuticallyeffective amount of the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, the pharmaceuticalcomposition the medicament, or the therapeutic combination product ofthe invention is to be administered for an acute treatment.

In one embodiment the therapeutically effective amount of the bacteriumof the invention and/or a variant thereof administered per day isranging from about 1·10² to about 1·10¹⁵ cfu/day, preferably from about1·10⁵ to about 1·10¹² cfu/day, more preferably from about 1·10⁸ to about1·10¹⁰ cfu/day, and even more preferably from about 1·10⁹ to about1·10¹⁰ cfu/day. In one embodiment the therapeutically effective amountof the bacterium of the invention and/or a variant thereof administeredper day is ranging from about 1·10⁷ to about 1·10¹⁰ cfu/day, preferablyfrom about 1·10⁸ to about 1·10⁹ cfu/day.

In one embodiment the therapeutically effective amount of the bacteriumof the invention administered per day is ranging from about 1·10⁶ toabout 1·10¹⁰ cfu/day, preferably from about 1·10⁸ to about 1·10¹⁰cfu/day, more preferably from about 1·10⁹ to about 1·10¹⁰ cfu/day.

In one embodiment the therapeutically effective amount of the bacteriumof the invention administered per day is ranging from about 1·10⁶ toabout 1·10¹¹ cfu/day, preferably from about 1·10⁸ to about 1·10¹¹cfu/day, more preferably from about 1·10¹⁰ to about 1·10¹¹ cfu/day.

In one embodiment the therapeutically effective amount of the bacteriumof the invention and/or a variant thereof administered per day isranging from about 1·10² to about 1·10¹⁵ cells/day, preferably fromabout 1·10⁵ to about 1·10¹² cells/day, more preferably from about 1·10⁸to about 1·10¹¹ cells/day, and even more preferably from about 1·10⁹ toabout 1·10¹⁰ cells/day. In one embodiment the therapeutically effectiveamount of the bacterium of the invention and/or a variant thereofadministered per day is ranging from about 1·10⁸ to about 1·10¹¹cells/day, preferably from about 5·10⁸ to about 5·10¹⁰ cells/day.

In one embodiment the therapeutically effective amount of the bacteriumof the invention administered per day is ranging from about 1·10⁶ toabout 1·10¹⁰ cells/day, preferably from about 1·10⁸ to about 1·10¹⁰cells/day, more preferably from about 1·10⁹ to about 1·10¹⁰ cells/day.

In one embodiment the therapeutically effective amount of the bacteriumof the invention administered per day is ranging from about 1·10⁶ toabout 1·10¹¹ cells/day, preferably from about 1·10⁸ to about 1·10¹¹cells/day, more preferably from about 1·10¹⁰ to about 1·10¹¹ cells/day.

In one embodiment the therapeutically effective amount of fragment ofthe bacterium of the invention administered per day corresponds to anamount of bacterium of the invention and/or a variant thereof rangingfrom about 1·10² to about 1·10¹⁵ cfu/day, preferably from about 1·10⁵ toabout 1·10¹² cfu/day, more preferably from about 1·10⁸ to about 1·10¹⁰cfu/day, and even more preferably from about 1·10⁹ to about 1·10¹⁰cfu/day. In one embodiment the therapeutically effective amount offragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention and/or a variantthereof ranging from about 1·10⁷ to about 1·10¹⁰ cfu/day, preferablyfrom about 1·10⁸ to about 1·10⁹ cfu/day.

In one embodiment the therapeutically effective amount of fragment ofthe bacterium of the invention administered per day corresponds to anamount of bacterium of the invention ranging from about 1·10⁶ to about1·10¹⁰ cfu/day, preferably from about 1·10⁸ to about 1·10¹⁰ cfu/day,more preferably from about 1·10⁹ to about 1·10¹⁰ cfu/day.

In one embodiment the therapeutically effective amount of fragment ofthe bacterium of the invention administered per day corresponds to anamount of bacterium of the invention ranging from about 1·10⁶ to about1·10¹¹ cfu/day, preferably from about 1·10⁸ to about 1·10¹¹ cfu/day,more preferably from about 1·10¹⁰ to about 1·10¹¹ cfu/day.

In one embodiment the therapeutically effective amount of fragment ofthe bacterium of the invention administered per day corresponds to anamount of bacterium of the invention and/or a variant thereof rangingfrom about 1·10² to about 1·10¹⁵ cells/day, preferably from about 1·10⁵to about 1·10¹² cells/day, more preferably from about 1·10⁸ to about1·10¹¹ cells/day, and even more preferably from about 1·10⁹ to about1·10¹⁰ cells/day. In one embodiment the therapeutically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention and/or a variantthereof ranging from 1·10⁸ to about 1·10¹¹ cells/day, preferably fromabout 5·10⁸ to about 5·10¹⁰ cells/day.

In one embodiment the therapeutically effective amount of fragment ofthe bacterium of the invention administered per day corresponds to anamount of bacterium of the invention ranging from about 1·10⁶ to about1·10¹⁰ cells/day, preferably from about 1·10⁸ to about 1·10¹⁰ cells/day,more preferably from about 1·10⁹ to about 1·10¹⁰ cells/day.

In one embodiment the therapeutically effective amount of fragment ofthe bacterium of the invention administered per day corresponds to anamount of bacterium of the invention ranging from about 1·10⁶ to about1·10¹¹ cells/day, preferably from about 1·10⁸ to about 1·10¹¹ cells/day,more preferably from about 1·10¹⁰ to about 1·10¹¹ cells/day.

The present invention also relates to the nutraceutical use of at leastone bacterium of the invention and/or a variant thereof and/or afragment thereof for obtaining a physiological benefit, improvingwell-being or alleviating a discomfort in a subject.

The present invention further relates to the cosmetic use of at leastone bacterium of the invention and/or a variant thereof and/or afragment thereof to ameliorate the perception, by the subject and/or byothers, of the appearance of the subject.

In one embodiment, the composition of the invention is in the form of afood additive, drink additive, dietary supplement, nutritional product,medical food or nutraceutical composition.

The present invention further relates to a nutraceutical compositioncomprising the composition according to the invention and at least onenutraceutically acceptable excipient.

The present invention further relates to a cosmetic compositioncomprising the composition according to the invention and at least onecosmetically acceptable excipient.

In the nutraceutical or cosmetic composition of the present invention,the bacterium of the invention and/or a variant thereof and/or fragmentthereof, alone or in combination with another active principle, can beadministered in a unit administration form, as a mixture withconventional supports, to animals and human beings. Suitable unitadministration forms comprise oral-route forms such as tablets, gelcapsules, powders, granules and oral suspensions or solutions,sublingual and buccal administration forms, aerosols, implants,subcutaneous, transdermal, topical, intraperitoneal, intramuscular,intravenous, subdermal, intrathecal, intranasal and rectaladministration forms.

In one embodiment, the nutraceutical or cosmetic composition accordingto the present invention can be used as dietary supplement to food andbeverages.

The nutraceutical or cosmetic composition according to the presentinvention may further contain protective hydrocolloids (such as gums,proteins, modified starches), binders, film-forming agents,encapsulating agents/materials, wall/shell materials, matrix compounds,coatings, emulsifiers, surface active agents, solubilizing agents (oils,fats, waxes, lecithins etc.), adsorbents, carriers, fillers,co-compounds, dispersing agents, wetting agents, processing aids(solvents), flowing agents, taste-masking agents, weighting agents,jellifying agents, gel-forming agents, antioxidants and antimicrobials.

Moreover, a multi-vitamin and mineral supplement may be added to thenutraceutical or cosmetic compositions of the present invention toobtain an adequate amount of an essential nutrient, which is missing insome diets. The multi-vitamin and mineral supplement may also be usefulfor disease prevention and protection against nutritional losses anddeficiencies due to lifestyle patterns.

The nutraceutical or cosmetic compositions according to the presentinvention may be in any galenic form that is suitable for administeringto the body, especially in any form that is conventional for oraladministration,

In one embodiment, the nutraceutical or cosmetic composition of theinvention is to be orally administered.

Examples of formulations adapted to oral administration include, but arenot limited to, solid forms, liquid forms and gels. Examples of solidforms adapted to oral administration include, but are not limited to,pill, tablet, capsule, soft gelatine capsule, hard gelatine capsule,dragees, granules, caplet, compressed tablet, cachet, wafer,sugar-coated pill, sugar coated tablet, or dispersing/or disintegratingtablet, powder, solid forms suitable for solution in, or suspension in,liquid prior to oral administration and effervescent tablet. Examples ofliquid form adapted to oral administration include, but are not limitedto, solutions, suspensions, drinkable solutions, elixirs, sealed phial,potion, drench, syrup, liquor and sprays.

Other examples of solid forms adapted to oral administration include,but are not limited to, (additives/supplements for) food or feed, foodor feed premix, fortified food or feed, tablets, pills, granules,dragees, capsules and effervescent formulations, such as powders andtablets.

Other examples of liquid forms adapted to oral administration include,but are not limited to, solutions, emulsions or suspensions such as,e.g., beverages, pastes and oily suspensions. The pastes may beincorporated in hard- or soft-shell capsules, whereby the capsulesfeature, e.g., a matrix of (fish, swine, poultry, cow) gelatine, plantproteins or ligninsulfonate.

In one embodiment of the invention, the composition, the nutraceuticalcomposition or the cosmetic composition of the invention is in the formof a nutritional composition, i.e., comprises liquid or solid food, feedor drinking water. In one embodiment of the invention, the composition,the nutraceutical composition or the cosmetic composition of theinvention is a food product, such as, for example, dairy products, dairydrinks, yogurt, fruit or vegetable juice or concentrate thereof,powders, malt or soy or cereal based beverages, breakfast cereal such asmuesli flakes, fruit and vegetable juice powders, cereal and/orchocolate bars, confectionary, spreads, flours, milk, smoothies,confectionary, milk product, milk powder, reconstituted milk, culturedmilk, yoghurt, drinking yoghurt, set yoghurt, drink, dairy drink, milkdrink, chocolate, gels, ice creams, cereals, reconstituted fruitproducts, snack bars, food bars, muesli bars, spreads, sauces, dips,dairy products including yoghurts and cheeses, drinks including dairyand non-dairy based drinks, sports supplements including dairy andnon-dairy based sports supplements.

Examples of food are dairy products including, but not limited to,margarines, spreads, butter, cheese, sausage, sauce, yoghurts,milk-drinks, ice cream, gums, chewing gum, gummi candy, taffy, caramelcandy, fudge and hard candy.

Examples of fortified food include, but are not limited to, sweet corn,bread, cereal bars, bakery items, such as cakes, pies and cookies, andpotato chips or crisps.

Beverages encompass non-alcoholic and alcoholic drinks as well as liquidpreparations to be added to drinking water and liquid food.Non-alcoholic drinks include, but are not limited to, soft drinks,sports drinks, energy drinks, fruit juices, lemonades, sodas, teas andmilk-based drinks. Liquid foods include, but are not limited to, soupsand dairy products.

In one embodiment, the composition, the nutraceutical composition or thecosmetic composition of the invention further comprises additionalprobiotic strains or species, such as, for example, bacterial probioticstrains or species; prokaryotes probiotics other than bacteria; orfungal strains or species, preferably yeast strains or species. In oneembodiment, said additional probiotic strains or species are selectedfrom those naturally present in the gut of the subject, preferably inthe human gut, more preferably in the gut of substantially healthy humansubjects. Examples of probiotic strains are listed hereinabove.

In one embodiment, the composition, nutraceutical composition or thecosmetic composition of the invention further comprises a prebiotic.Examples of prebiotic compounds are listed hereinabove.

The present invention also relates to a nutraceutical or cosmeticcombination product for its separate, simultaneous or sequentialadministration.

As used herein the term “nutraceutical or cosmetic combination product”(that may also be referred to as a nutraceutical or cosmetic kit ofparts) refers to a product comprising or consisting of at least the 2following parts: a first part comprising (preferably in anutraceutically or cosmetically effective affective amount) a bacteriumof the invention and/or a variant thereof, a composition according tothe invention, a nutraceutical composition according to the invention,or a cosmetic composition according to the invention and a second partcomprising a composition comprising a least one probiotic and/or atleast one prebiotic.

In one embodiment, the at least two parts of a nutraceutical or cosmeticcombination product according to the invention are administeredsimultaneously. In another embodiment, the at least two parts of thenutraceutical or cosmetic combination product according to the inventionare administered at a different time. In another embodiment, the atleast two parts of the nutraceutical or cosmetic combination productaccording to the invention are administered sequentially.

In one embodiment, the at least two parts of the nutraceutical orcosmetic combination product according to the invention are administeredusing different administration routes. (such as, for example, one partby oral administration, and the second one by injection). In anotherembodiment, the at least two parts of the nutraceutical or cosmeticcombination product according to the invention are administered usingthe same administration route (such as, for example, oral administrationor injection).

The present invention further relates to non-therapeutic methods forimproving well-being, obtaining a physiological, and/or obtaining acosmetic benefit in a subject, wherein said methods compriseadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or fragments thereof, or the composition,nutraceutical composition or combination product, or cosmeticcomposition or combination product of the invention.

The present invention thus relates to the nutraceutical or cosmetic useof the bacterium of the invention and/or a variant thereof and/orfragments thereof.

In one embodiment, the method of the invention comprises administering acosmetically effective amount of the bacteria of the invention and/orfragment thereof, of the composition or the cosmetic composition of theinvention to the subject.

In one embodiment, the method of the invention comprises administering anutraceutically effective amount of the bacteria of the invention and orfragment thereof, or of the composition or the nutraceutical compositionor combination product of the invention to the subject.

As used herein, the term “physiological benefit” refers to an increasein the efficiency of a physiological function in a subject, or a partialor complete alleviation of a discomfort in a subject.

As used herein, the term “well-being” refers to an amelioration of theperception by the subject of his own health status.

As used herein, the term “cosmetic benefit” refers to an amelioration ofthe perception, by the subject and/or by others, of the appearance ofthe subject.

In one embodiment, the method of the invention is a method for improvingwell-being in a subject. The present invention thus relates to the useof the bacterium of the invention and/or a variant thereof and/orfragments thereof for improving well-being in a subject.

In one embodiment, the method of the invention is a method for promotingweight loss in a subject. The present invention thus relates to the useof the bacterium of the invention and/or a variant thereof and/orfragments thereof for promoting weight loss in a subject.

In one embodiment, the method of the invention is a method fordecreasing food intake in a subject. The present invention thus relatesto the use of the bacterium of the invention and/or a variant thereofand/or fragments thereof for decreasing food intake in a subject.

In one embodiment, the method of the invention is a method forincreasing muscle mass in a subject. The present invention thus relatesto the use of the bacterium of the invention and/or a variant thereofand/or fragments thereof for increasing muscle mass in a subject.

In one embodiment, the method of the invention is a method fordecreasing fat mass in a subject. The present invention thus relates tothe use of the bacterium of the invention and/or a variant thereofand/or fragments thereof for decreasing fat mass in a subject. In oneembodiment, the bacterium of the invention and/or a variant thereofand/or fragments thereof decreases abnormal fat accumulation, alteredlipolysis, and high-fat storage. In one embodiment, the bacterium of theinvention and/or a variant thereof and/or fragments thereof increaseslipolysis.

In one embodiment, the method of the invention is a method forincreasing satiety in a subject. The present invention thus relates tothe use of the bacterium of the invention and/or a variant thereofand/or fragments thereof for increasing satiety in a subject.

In one embodiment, the method of the invention is a method fordecreasing the weight gain associated with food intake in a subject. Thepresent invention thus relates to the use of the bacterium of theinvention and/or a variant thereof and/or fragments thereof fordecreasing the weight gain associated with food intake in a subject.

In one embodiment, the method of the invention is a method fordecreasing the intestinal absorption associated with food intake in asubject. The present invention thus relates to the use of the bacteriumof the invention and/or a variant thereof and/or fragments thereof fordecreasing the intestinal absorption associated with food intake in asubject.

In one embodiment, the subject is substantially healthy, in particularin respect to disorder related to the gut microbiota.

In one embodiment, the subject is not obese. In one embodiment, thesubject has a BMI lower than about 40, preferably lower than about 35,more preferably lower than about 30. In one embodiment, the subject isnot overweight. In one embodiment the subject has a BMI lower than aboutthan about 30, more preferably lower than about 25.

In one embodiment, the bacterium of the invention and/or a variantthereof and/or fragments thereof, the composition, the nutraceuticalcomposition or combination product or the cosmetic composition orcombination product of the invention is to be administered at a dosedetermined by the skilled artisan and personally adapted to eachsubject.

In addition, the specific nutraceutically or cosmetically effectiveamount for any particular subject will depend upon a variety of factorsincluding the specific composition employed, the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, the duration of the treatment; drugs used incombination or coincidental with the composition of the invention; andlike factors well known in the medical, nutraceutical and cosmetic arts.

In one embodiment, a nutraceutically or cosmetically effective amount ofthe bacterium of the invention and/or a variant thereof and/or fragmentsthereof, or of the composition, the nutraceutical composition orcombination product, or the cosmetic composition or combination productof the invention is to be administered at least once a day, at leasttwice a day, at least three times a day.

In one embodiment, a nutraceutically or cosmetically effective amount ofthe bacterium of the invention and/or a variant thereof and/or fragmentsthereof, or of the composition, the nutraceutical composition orcombination product, or the cosmetic composition or combination productof the invention is to be administered every two, three, four, five, sixdays.

In one embodiment, a nutraceutically or cosmetically effective amount ofthe bacterium of the invention and/or a variant thereof and/or fragmentsthereof, or of the composition, the nutraceutical composition orcombination product, or the cosmetic composition or combination productof the invention is to be administered twice a week, every week, everytwo weeks, once a month.

In one embodiment, a nutraceutically or cosmetically effective amount ofthe bacterium of the invention and/or a variant thereof and/or fragmentsthereof, or of the composition, the nutraceutical composition orcombination product, or the cosmetic composition or combination productof the invention is to be administered every month, every two months,every three months, every four months, every five months, every sixmonths, once a year.

In one embodiment, a nutraceutically or cosmetically effective amount ofthe bacterium of the invention and/or a variant thereof and/or fragmentsthereof, or of the composition, the nutraceutical composition orcombination product, or the cosmetic composition or combination productof the invention is to be administered for a period of time of about oneday, two days, three days, four days, five days, six days, a week, twoweeks, three weeks, a month, two months, three months, six months, ayear, or over longer periods such as, e.g., for several years or for therest of the life of the subject.

In one embodiment, when a nutraceutical or cosmetic combination productof the invention is to be administered to the subject, each part of saidcombination product may be administered at a different frequency and fora different period of time.

In one embodiment, a nutraceutically or cosmetically effective amount ofthe bacterium of the invention and/or a variant thereof and/or fragmentsthereof, or of the composition, the nutraceutical composition orcombination product or the cosmetic composition or combination productof the invention is to be administered until the desired physiologicalor cosmetic benefit has been achieved.

Example of physiological or cosmetic benefits include, but is notlimited to, an improvement of well-being, weight loss, increase inmuscle mass, reduction of fat mass, reduction of food intake, increasein satiety, decrease in the weight gain associated with food intake,decrease of the intestinal absorption associated with food intake.

In one embodiment the nutraceutically or cosmetically effective amountof the bacterium of the invention and/or a variant thereof administeredper day is ranging from about 1·10² to about 1·10¹⁵ cfu/day, preferablyfrom about 1·10⁵ to about 1·10¹² cfu/day, more preferably from about1·10⁸ to about 1·10¹⁰ cfu/day, and even more preferably from about 1·10⁹to about 1·10¹⁰ cfu/day. In one embodiment the nutraceutically orcosmetically effective amount of the bacterium of the invention and/or avariant thereof administered per day is ranging from about 1·10⁷ toabout 1·10¹¹ cfu/day, preferably from about 1·10⁸ to about 1·10¹⁰cfu/day.

In one embodiment the nutraceutically or cosmetically effective amountof the bacterium of the invention administered per day is ranging fromabout 1·10⁶ to about 1·10¹⁰ cfu/day, preferably from about 1·10⁸ toabout 1·10¹⁰ cfu/day, more preferably from about 1·10⁹ to about 1·10¹⁰cfu/day.

In one embodiment nutraceutically or cosmetically effective amount ofthe bacterium of the invention administered per day is ranging fromabout 1·10⁶ to about 1·10¹¹ cfu/day, preferably from about 1·10⁸ toabout 1·10¹¹ cfu/day, more preferably from about 1·10¹⁰ to about 1·10¹¹cfu/day.

In one embodiment the nutraceutically or cosmetically effective amountof the bacterium of the invention and/or a variant thereof administeredper day is ranging from about 1·10² to about 1·10¹⁵ cells/day,preferably from about 1·10⁵ to about 1·10¹² cells/day, more preferablyfrom about 1·10⁸ to about 1·10¹⁰ cells/day, and even more preferablyfrom about 1·10⁹ to about 1·10¹⁰ cells/day. In one embodiment thenutraceutically or cosmetically effective amount of the bacterium of theinvention and/or a variant thereof administered per day is ranging fromabout 1·10⁸ to about 1·10¹¹ cells/day, preferably from about 5·10⁸ toabout 5·10¹⁰ cells/day.

In one embodiment the nutraceutically or cosmetically effective amountof the bacterium of the invention administered per day is ranging fromabout 1·10⁶ to about 1·10¹⁰ cells/day, preferably from about 1·10⁸ toabout 1·10¹⁰ cells/day, more preferably from about 1·10⁹ to about 1·10¹⁰cells/day.

In one embodiment the nutraceutically or cosmetically effective amountof the bacterium of the invention administered per day is ranging fromabout 1·10⁶ to about 1·10¹¹ cells/day, preferably from about 1·10⁸ toabout 1·10¹¹ cells/day, more preferably from about 1·10¹⁰ to about1·10¹¹ cells/day.

In one embodiment the nutraceutically or cosmetically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention and/or a variantthereof ranging from about 1·10² to about 1·10¹⁵ cfu/day, preferablyfrom about 1·10⁵ to about 1·10¹² cfu/day, more preferably from about1·10⁸ to about 1·10¹⁰ cfu/day, and even more preferably from about 1·10⁹to about 1·10¹⁰ cfu/day. In one embodiment the nutraceutically orcosmetically effective amount of fragment of the bacterium of theinvention administered per day corresponds to an amount of bacterium ofthe invention and/or a variant thereof ranging from about 1·10⁷ to about1·10¹¹ cfu/day, preferably from about 1·10⁸ to about 1·10¹⁰ cfu/day.

In one embodiment the nutraceutically or cosmetically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹⁰ cfu/day, preferably from about 1·10⁸ toabout 1·10¹⁰ cfu/day, more preferably from about 1·10⁹ to about 1·10¹⁰cfu/day.

In one embodiment the nutraceutically or cosmetically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹¹ cfu/day, preferably from about 1·10⁸ toabout 1·10¹¹ cfu/day, more preferably from about 1·10¹⁰ to about 1·10¹¹cfu/day.

In one embodiment the nutraceutically or cosmetically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention and/or a variantthereof ranging from about 1·10² to about 1·10¹⁵ cells/day, preferablyfrom about 1·10⁵ to about 1·10¹² cells/day, more preferably from about1·10⁸ to about 1·10¹¹ cells/day, and even more preferably from about1·10⁹ to about 1·10¹⁰ cells/day. In one embodiment the nutraceuticallyor cosmetically effective amount of fragment of the bacterium of theinvention administered per day corresponds to an amount of bacterium ofthe invention and/or a variant thereof ranging from about 1·10⁸ to about1·10¹¹ cells/day, preferably from about 5·10⁸ to about 5·10¹⁰ cells/day.

In one embodiment the nutraceutically or cosmetically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹⁰ cells/day, preferably from about 1·10⁸ toabout 1·10¹⁰ cells/day, more preferably from about 1·10⁹ to about 1·10¹⁰cells/day.

In one embodiment the nutraceutically or cosmetically effective amountof fragment of the bacterium of the invention administered per daycorresponds to an amount of bacterium of the invention ranging fromabout 1·10⁶ to about 1·10¹¹ cells/day, preferably from about 1·10⁸ toabout 1·10¹¹ cells/day, more preferably from about 1·10¹⁰ to about1·10¹¹ cells/day.

The present invention relates to a method for treating a disorderrelated to the gastrointestinal microbiota in a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention also relates to a method for restoring and/orenhancing function associated with the gut microbiota in a subject,comprising administering to the subject at least one bacterium of theinvention and/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for decreasing glucoseintolerance and/or insulin resistance in a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for restoring the normalproportion of the bacteria of the invention present in the gutmicrobiota of a subject in need thereof, comprising administering to thesubject at least one bacterium of the invention and/or a variant thereofand/or at least one fragment thereof. In one embodiment, said normalproportion corresponds to the proportion observed in a substantiallyhealthy subject. In one embodiment, said normal proportion ranges fromabout 0.1% to about 5% of the total bacteria found in the feces of thesubject, preferably from about 0.5% to about 4%, more preferably fromabout 0.8% to about 3% In one embodiment, the normal proportion in asubstantially healthy subject is about 2.45% of the total bacteria foundin the feces of the subject. In one embodiment, said normal proportionis at least 0.5%, preferably at least 0.6%, 0.7%, 0.8%, 0.85%, 0.90%,0.95%, 1%, 1.05%, 1.10%, 1.15%, 1.20%, 1.25%, 1.30%, 1.35%, 1.40%,1.45%, more preferably at least 1.50%, 1.55%, 1.60%, 1.65%, 1.70%,1.75%, 1.80%, 1.85%, 1.90%, 2%, 2.05%, 2.10%, 2.15%, 2.20%, 2.25%,2.30%, 2.35%, 2.40% or more of the total bacteria found in the feces ofthe substantially healthy subject.

Techniques to determine the presence and/or proportion of the bacteriaof the invention are known to the skilled artisan and include withoutlimitation: PCR, qPCR, hybridization techniques (FISH, Northern Blot),bacteria identification kit, and DNA or RNA sequencing and techniques ofimmunodetection.

The present invention relates to a method for decreasing thepermeability of the gut intestinal barrier in a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for increasing the expressionlevel of genes involved in the establishment and/or maintenance of theepithelial barrier, such as, for example genes coding for mucins(muc—including but not limited to mucin 2), occludins, claudins(cldn—including but not limited to claudin 3, 4, 7, 15 and 23),defensins (defa—including but not limited to defensin 5, 17, 21, 22, 24,30, 34), Regenerating islet-derived protein (reg—including but notlimited to regenerating islet-derived protein 1, 3a, 3b and 3g)junctional adhesion molecules (JAM), E-cadherin, catenins, nectin,afadin, zonulin and zonula occludens (ZO)-1, ZO-2 and ZO-3, wherein themethod of the invention comprises administering to the subject at leastone bacterium of the invention and/or at least one fragment thereof.

The present invention relates to a method for increasing the secretionof mucus by the intestinal epithelium, wherein the method of theinvention comprises administering to the subject at least one bacteriumof the invention and/or a variant thereof and/or at least one fragmentthereof.

The present invention relates to a method for increasing the level ofproglucagon and/or glucagon-like peptide 1 (GLP-1) and/or glucagon-likepeptide 2 (GLP-2) in a subject, comprising administering to the subjectat least one bacterium of the invention and/or a variant thereof and/orat least one fragment thereof.

The present invention relates to a method for decreasing fibrosis of theadipose tissues in a subject, comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

The present invention relates to a method for decreasing the expressionof genes involved in fibrosis in the adipose tissues of a subject,preferably genes selected from the group comprising, or consisting of,Thbs2, Col6a2, S100a6, Myoc, Col1a2, Col1a1, Col5a2, Col3a1, Ntn1,Ctsc2, Serpinf1, Adamts4, Col6a1, Anxa1, Nid2, Cilp, Lum and Mmp14,comprising administering to the subject at least one bacterium of theinvention and/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for decreasing the expressionof genes involved in fibrosis in the brown adipose tissues of a subject,preferably genes selected from the group comprising, or consisting of,Thbs2, Col6a2, S100a6, Myoc, Col1a2, Col1a1, Col5a2, Col3a1, Ntn1,Ctsc2, Serpinf1, Adamts4, Col6a1, Anxa1, Nid2, Cilp, Lum, Mmp14, andCol10a, comprising administering to the subject at least one bacteriumof the invention and/or a variant thereof and/or at least one fragmentthereof.

The present invention relates to a method for decreasing the expressionof genes involved in fibrosis in the subcutaneous adipose tissues of asubject, preferably genes selected from the group comprising, orconsisting of, Thbs2, Adamts12, Lum, Cela1, S100a6, Cilp, Ctsc2,Serpinf1, Anxa1, Mmp14, Col1a2, Col3a1, Col5a2, Col6a1, Col6a2, Col1a1,Myoc, Nid2, Ntn1 and Adamts4, comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

The present invention relates to a method for decreasing inflammation ofthe adipose tissues, preferably brown adipose tissues, in a subject,comprising administering to the subject at least one bacterium of theinvention and/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for decreasing the expressionof genes involved in the inflammation of the adipose tissues, preferablybrown adipose tissues, of a subject, preferably genes selected from thegroup comprising, or consisting of, Naip6, Anxa1, Lbp, Ccl6, Pycard,F2rl1, Ccl9, Smpdl3b, Cela1, Tnfrsf1b, Cd5l, Ptafr, C3ar1, Pf4, Loxl3,Ccr5, Saa3, Ccr1, Adra2a, Tlr13, Ccl2, Alox5, Fcgr1, Ccl7, Ccl8, Ptgs2,Havcr2, Relb2, Ticam2, Stab1, Themis2_2, Tlr11, Ptger2, Orm2, Cxcr3,Pxk1, Pelb1, Nlrp10, Ccl12, Ppbp, Cd180, C,cl3, Ptgir2, Ptgir1, Chst1_2,Adma8, Nrros, Ptger1 and Themis2_1, comprising administering to thesubject at least one bacterium of the invention and/or a variant thereofand/or at least one fragment thereof.

The present invention relates to a method for reducing diet-inducedfasting hyperglycemia in a subject, comprising administering to thesubject at least one bacterium of the invention and/or a variant thereofand/or at least one fragment thereof.

The present invention relates to a method for increasing energyexpenditure of a subject comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

The present invention relates to a method for decreasing energyintestinal absorption of a subject comprising administering to thesubject at least one bacterium of the invention and/or a variant thereofand/or at least one fragment thereof.

When sharing the same environment interactions between microorganismsmay lead to competitive and/or collaborative effect.

The present invention thus further relates to a method for activatingthe growth and/or biological activity of other microorganisms of the gutmicrobiota in a subject, comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

The present invention also relates to a method for inhibiting the growthand/or biological activity of other microorganisms of the gut microbiotain a subject, comprising administering to the subject at least onebacterium of the invention and/or a variant thereof and/or at least onefragment thereof.

The present invention also relates to a method for assisting in defenseagainst exogenous pathogenic bacteria in a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for increasing the productionof antibacterial compound(s) in a subject, comprising administering tothe subject at least one bacterium of the invention and/or a variantthereof and/or at least one fragment thereof.

The present invention relates to a method for increasing the productionof defensin in a subject, comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

Bacteria of the gut microbiota participate in the metabolism of theirhost by participating in the digestion and producing vitamins.

The present invention thus further relates to a method for assistingdigestion and/or producing vitamins in a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

Butyrate, produced by anaerobe bacteria of the gut microbiota hasnumerous beneficial effects on the host health on energy metabolism andrelated metabolic diseases. it also has as an immunomodulatory,antimicrobial and anticarcinogenic effect. Bacteria belonging to theclostridial cluster IV, as the bacterium of the invention are majorbutyrate producer. Beyond the sole role of butyrate, other short-chainfatty acids (SCFA), such as formate, acetate, isobutyrate, propionateisovalerate and valerate, have beneficial effect on the metabolism andimmunity of their host.

The present invention thus further relates to a method for increasingthe production of SCFA in the gut of a subject, comprising administeringto the subject at least one bacterium of the invention and/or a variantthereof and/or at least one fragment thereof.

The present invention also relates to a method for increasing theproduction of butyrate, isobutyrate, butyric acid and/or isobutyric acidin the gut of a subject, comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

The present invention relates to a method for increasing the productionof acetate and/or acetic acid in the gut of a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for increasing the productionof propionate, isopropionate, propionic acid and/or iso-propionic acidin the gut of a subject, comprising administering to the subject atleast one bacterium of the invention and/or a variant thereof and/or atleast one fragment thereof.

The present invention relates to a method for deceasing the expressionof genes involved in the inflammatory response in the adipose tissues,preferably brown and/or subcutaneous adipose tissues, of a subject,comprising administering to the subject at least one bacterium of theinvention and/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for deceasing the expressionof genes involved in the inflammatory response in the adipose tissues,preferably brown and/or subcutaneous adipose tissues, of a subject,comprising administering to the subject at least one bacterium of theinvention and/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for deceasing the expressionof genes involved in fibrosis in the adipose tissues, preferably brownand/or subcutaneous adipose tissues, of a subject, comprisingadministering to the subject at least one bacterium of the inventionand/or a variant thereof and/or at least one fragment thereof.

The present invention relates to a method for increasing the activationof the Toll-like receptor 2 (TLR2) receptor in the intestine of asubject, comprising administering to the subject at least one bacteriumof the invention and/or a variant thereof and/or at least one fragmentthereof. In one embodiment, the bacterium of the invention and/or avariant thereof and/or fragments thereof increases the activation of theToll-like receptor 2 (TLR2) receptor in the intestinal epithelial cellsof a subject.

The present invention relates to a method for increasing the productionof valerate, isovalerate, valeric acid and/or isovaleric acid in the gutof a subject, comprising administering to the subject at least onebacterium of the invention and/or a variant thereof and/or at least onefragment thereof.

In one embodiment, the therapeutic methods of the invention compriseadministering the composition, pharmaceutical composition or medicamentof the invention to the subject. In one embodiment, a therapeuticallyeffective amount of the at least one bacterium of the invention and/or avariant thereof and/or at least one fragment thereof is administered tothe subject.

In one embodiment, the non-therapeutic methods of the invention compriseadministering the composition, nutraceutical composition or cosmeticcomposition of the invention to the subject. In one embodiment, acosmetically or nutraceutically effective amount of the at least onebacterium of the invention and/or a variant thereof and/or at least onefragment thereof is administered to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows scanning electron micrographs of cells of strain J115 inexponential phase. (a) 50 000 magnification, scale bar=1 μm. (b) 10 000magnification, scale bar=2 μm.

FIG. 2 shows phylogenetic trees based on 16S rRNA gene sequences,showing the connections between strain J115 (Dysosmobacter welbionis),Oscillibacter. ruminantium JCM 18333^(T) (=GH1T=KCTC 15176=NBRC 108824)Oscillibacter valericigenes DSM 18026^(T) (Sjm18-20T=NBRC 101213),Oscillospira guilliermondii clones and other related taxa. GenBankaccession numbers are shown in parentheses. Bootstrap values based on1000 replicates are indicated on branch points. Bars, 0.02 substitutionsper nucleotide position. (a) Neighbor-joining phylogenetic tree, (b)Maximum-likelihood phylogenetic tree.

FIG. 3 shows phylogenetic trees of the MLSA based on the concatenatedsequences of twelve protein-coding genes, showing the connectionsbetween strain J115 (Dysosmobacter welbionis), Oscillibacter.ruminantium JCM 18333^(T) (=GH1T=KCTC 15176=NBRC 108824) Oscillibactervalericigenes DSM 18026^(T) (Sjm18-20^(T)=NBRC 101213) and other relatedtaxa. GenBank accession numbers of the complete genomes used to retrievethe genes sequences are shown in parentheses. Bootstrap values based on1000 replicates are indicated on branch points. The twelve genesincluded in the MLSA are RNA polymerase sporulation specific sigmafactor SigE (sigE), phosphoribosylformylglycinamidine cyclo-ligase(purM), argininosuccinate synthase (ass), aspartokinase (lysC),phosphate starvation-inducible protein (phoH), catabolite repressionHPr-like protein (crh), chaperone groEL (groEL), tRNA modificationGTPase MnmE (thdF), translation initiation factor IF-2 (infB), proteinrecA (recA), RNA polymerase sigma factor RpoD (rpoD) and DNA gyrasesubunit B (gyrB). Bars, 0.05 substitutions per nucleotide position. (A)Neighbor-joining phylogenetic tree, (B) Maximum-likelihood phylogenetictree.

FIG. 4 shows an unweighted pair group method with arithmetic mean(UPGMA) phylogenetic tree of the average nucleotide identity (ANI) basedon the whole genomes, showing the connections between strain J115(Dysosmobacter welbionis), Oscillibacter. ruminantium JCM 18333^(T)(=GH1T=KCTC 15176=NBRC 108824) Oscillibacter valericigenes DSM 18026^(T)(Sjm18-20^(T)=NBRC 101213) and other related taxa. GenBank accessionnumbers of the complete genomes used are shown in parentheses.

FIG. 5 shows an UPGMA phylogenetic tree of the intergenome distances(GGD) based on the whole genomes, showing the connections between strainJ115 (Dysosmobacter welbionis), Oscillibacter ruminantium JCM 18333^(T)(=GH1T=KCTC 15176=NBRC 108824) Oscillibacter valericigenes DSM 18026^(T)(Sjm18-20^(T)=NBRC 101213) and other related taxa. GenBank accessionnumbers of the complete genomes used are shown in parentheses.

FIG. 6 is a graph showing body weight curves of mice treated by dailyoral gavage with Dysosmobacter welbionis J115 (10⁹ bacterial cellssuspended in 200 μL sterile anaerobic phosphate-carbonate buffer saline(PCBS)) and fed a high-fat diet (HFD-Dysosmobacter J115) or mice fed acontrol diet (Control) or a high-fat diet (HFD) and treated by dailyoral gavage with an equivalent volume of sterile anaerobicPCBS-carbonate for 8-weeks (n=10/group).

FIG. 7 is a graph showing weight gain measured for each group at the endof the 8-weeks treatment as described in FIG. 6. Data are shown asscatter dot plot with median. *: p<0.05, Kruskal-Wallis test followed bypairwise comparisons.

FIG. 8 is a graph showing the daily food intake per mouse, calculatedbased on weekly food intake of 2-mice cages during the 8-weeks treatmentas described in FIG. 6. Data are shown as scatter dot plot with median.**: p<0.01, Kruskal-Wallis test followed by pairwise comparisons.

FIG. 9 is a graph showing the daily amount of calories ingested permouse during weeks 6 and 7 of the 8-weeks treatment as described in FIG.6. Data are shown as scatter dot plot with median. *: p<0.05,Kruskal-Wallis test followed by pairwise comparisons.

FIG. 10 is a graph showing the daily amount of calories excreted (fecalenergy output) per mouse during weeks 6 and 7 of the 8-weeks treatmentas described in FIG. 6. Data are shown as scatter dot plot with median.*: p<0.05, Kruskal-Wallis test followed by pairwise comparisons.

FIG. 11 is a graph showing the daily amount of calories absorbed(absorbed energy) per mouse during weeks 6 and 7 of the 8-weekstreatment as described in FIG. 6. Data are shown as scatter dot plotwith median. *: p<0.05, Kruskal-Wallis test followed by pairwisecomparisons.

FIG. 12 is a graph showing the ratio of weight gain over the food intakeat the end of the 8-weeks treatment as described in FIG. 6. Data areshown as scatter dot plot with median.

FIG. 13 is a graph showing Proglucagon mRNA expression levels measuredin the terminal ileum at the end of the 8-weeks treatment as describedin FIG. 6. Data are shown as scatter dot plot with median. **: p<0.01,Kruskal-Wallis test followed by pairwise comparisons.

FIG. 14 is a histogram showing the short chain fatty acid (SCFA)produced by Dysosmobacter welbionis strain J115 in vitro.

FIG. 15 is a graph showing occludin mRNA expression level measured inthe terminal ileum at the end of the 8-weeks treatment as described inFIG. 6. Data are shown as scatter dot plot with median. *: p<0.05,Kruskal-Wallis test followed by pairwise comparisons.

FIG. 16 is a graph showing the relative concentration of total GLP-1 inthe supernatant of GLUtag cells after 2 hours of exposition to differentconcentration of D. welbionis J1115^(T). Data are shown as scatter dotplot with median.

FIG. 17 is a graph showing the sum of the extracellular (supernatant)and intracellular relative concentration of GLP-1 in GLUtag cells after2 hours of exposition to different concentration of D. welbionisJ115^(T). Data are shown as scatter dot plot with median.

FIG. 18 is a graph showing the amount of dead and live cells of D.welbionis J115^(T) in the fresh and frozen suspensions administered permouse and per day.

FIG. 19 is a graph showing the body weight evolution of mice treated bydaily oral gavage with fresh and frozen suspensions of 5·10⁹ cells of D.welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group). Stars or (+) signs indicate significantdifferences (* or +: p<0.05; ** or ++: p<0.01; *** or +++: p<0.001)between two groups according to statistical analysis consisting ofone-way ANOVA followed by pairwise comparisons and Tukey correction. *indicate statistical significance between HFD and HFD-J115-fresh groupswhile + indicate statistical significance between HFD andHFD-J115-frozen groups.

FIG. 20 is a graph showing the total body weight gain of mice treated bydaily oral gavage with fresh and frozen suspensions of 5·10⁹ D.welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group), measured at the end of the 13-weeks period.Stars indicate significant differences (*: p<0.05) between two groupsaccording to statistical analysis consisting of one-way ANOVA followedby pairwise comparisons and Tukey correction.

FIG. 21 is a graph showing the daily food intake per mouse and per dayof mice treated by daily oral gavage with fresh and frozen suspensionsof 5·10⁹ D. welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group), calculated based of weekly food intake of 2mice cages over the 13-weeks period. Stars indicate significantdifferences (****: p<0.0001) between two groups according to statisticalanalysis consisting of one-way ANOVA followed by pairwise comparisonsand Tukey correction.

FIG. 22 is a graph showing the ingested energy (calories) per day andper mouse of mice treated by daily oral gavage with fresh and frozensuspensions of 5·10⁹ D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group), during weeks 6and 8 of the experiment. Data are shown as scatter dot plot with median.

FIG. 23 is a graph showing the fecal energy output (excreted calories)per day and per mouse of mice treated by daily oral gavage with freshand frozen suspensions of 5·10⁹ D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group), during weeks 6and 8 of the experiment. Data are shown as scatter dot plot with median.

FIG. 24 is a graph showing the absorbed energy (calories) per day andper mouse of mice treated by daily oral gavage with fresh and frozensuspensions of 5·10⁹ D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group), during weeks 6and 8 of the experiment. Daily oral gavage with fresh or frozen 5·10⁹cells of Dysosmobacter welbionis J115^(T). Data are shown as scatter dotplot with median.

FIG. 25 is a graph showing the ratio of weight gain over the food intakeof mice treated by daily oral gavage with fresh and frozen suspensionsof 5·10⁹ D. welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group), at the end of the 13 weeks experiment. Dataare shown as scatter dot plot with median. Stars indicate significantdifferences (**: p<0.01) between two groups according to statisticalanalysis consisting of one-way ANOVA followed by pairwise comparisonsand Tukey correction.

FIG. 26 is a graph showing the total fat mass evolution of mice treatedby daily oral gavage with fresh and frozen suspensions of 5·10⁹ cells ofD. welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group). Data are shown as scatter dot plot withmedian. Stars and plus signs indicate significant differences (* or +:p<0.05; ** or ++: p<0.01; *** or +++: p<0.001) between two groupsaccording to statistical analysis consisting of one-way ANOVA followedby pairwise comparisons and Tukey correction. * indicate statisticalsignificance between HFD and HFD-J115-fresh groups while + indicatestatistical significance between HFD and HFD-J115-frozen groups.

FIG. 27 is a graph showing the mesenteric, subcutaneous and epididymalfat pads weight of mice treated by daily oral gavage with fresh andfrozen suspensions of 5·10⁹ cells of D. welbionis J115^(T) and fed aHF-diet (HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed acontrol diet (Control) or a high-fat diet (HFD) and treated by dailyoral gavage with an equivalent volume of sterile trehalose 15% inanaerobic PBS-carbonate buffer saline for 13-weeks (n=12/group), at theend of the 13-weeks period. Data are shown as scatter dot plot withmedian. Stars indicate significant differences (*: p<0.05; **: p<0.01;***: p<0.001) between two groups according to statistical analysisconsisting of one-way ANOVA followed by pairwise comparisons and Tukeycorrection.

FIG. 28 is series of representative hematoxylin and eosin (H&E)-stainedpictures of subcutaneous adipose tissue (SCAT) of mice treated by dailyoral gavage with fresh and frozen suspensions of 5·10⁹ cells of D.welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group), at the end of the 13-weeks period.

FIG. 29 is a graph showing Adipocytes diameter (μm) distribution in theSCAT of mice treated by daily oral gavage with fresh and frozensuspensions of 5·10⁹ cells of D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group), at the end of the13-weeks period. Data are shown as scatter dot plot with median. Starsand plus signs indicate significant differences (* or +: p<0.05; ** or++: p<0.01; *** or +++: p<0.001) between two groups according tostatistical analysis consisting of one-way ANOVA followed by pairwisecomparisons and Tukey correction. * indicate statistical significancebetween HFD and HFD-J115-fresh groups while + indicate statisticalsignificance between HFD and HFD-J115-frozen groups.

FIG. 30 is series of representative hematoxylin and eosin (H&E)-stainedpictures of mesenteric adipose tissue (MAT) of mice treated by dailyoral gavage with fresh and frozen suspensions of 5·10⁹ cells of D.welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group), at the end of the 13-weeks period.

FIG. 31 is a graph showing adipocytes diameter (μm) distribution in theMAT of mice treated by daily oral gavage with fresh and frozensuspensions of 5·10⁹ cells of D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group), at the end of the13-weeks period. Data are shown as scatter dot plot with median. Starsand plus signs indicate significant differences (* or +: p<0.05; ** or++: p<0.01; *** or +++: p<0.001) between two groups according tostatistical analysis consisting of one-way ANOVA followed by pairwisecomparisons and Tukey correction. * indicate statistical significancebetween HFD and HFD-J115-fresh groups while + indicate statisticalsignificance between HFD and HFD-J115-frozen groups.

FIG. 32 is a graph showing plasma glucose (mg/dL) profile during oralglucose tolerance test (OGTT) of mice treated by daily oral gavage withfresh and frozen suspensions of 5·10⁹ cells of D. welbionis J115^(T) andfed a HF-diet (HFD-J115-fresh and HFD-J115-frozen, respectively) or micefed a control diet (Control) or a high-fat diet (HFD) and treated bydaily oral gavage with an equivalent volume of sterile trehalose 15% inanaerobic PBS-carbonate buffer saline for 13-weeks (n=12/group). Dataare shown as scatter dot plot with median. Stars and plus signs indicatesignificant differences (* or +: p<0.05; ** or ++: p<0.01; *** or +++:p<0.001) between two groups according to statistical analysis consistingof one-way ANOVA followed by pairwise comparisons and Tukeycorrection. * indicate statistical significance between HFD andHFD-J115-fresh groups while + indicate statistical significance betweenHFD and HFD-J115-frozen groups.

FIG. 33 is a graph showing plasma insulin concentration (μg/L) measured30 min before and 15 min after glucose administration during the OGTTduring oral glucose tolerance test (OGTT) of mice treated by daily oralgavage with fresh and frozen suspensions of 5·10⁹ cells of D. welbionisJ115^(T) and fed a HF-diet (HFD-J115-fresh and HFD-J115-frozen,respectively) or mice fed a control diet (Control) or a high-fat diet(HFD) and treated by daily oral gavage with an equivalent volume ofsterile trehalose 15% in anaerobic PBS-carbonate buffer saline for13-weeks (n=12/group). Data are shown as scatter dot plot with median.Stars indicate significant differences (*: p<0.05) between two groupsaccording to statistical analysis consisting of one-way ANOVA followedby pairwise comparisons and Tukey correction.

FIG. 34 is a graph showing fasting leptin concentration (ng/mL) in theplasma of mice treated by daily oral gavage with fresh and frozensuspensions of 5·10⁹ cells of D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group). Data are shown asscatter dot plot with median. Stars indicate significant differences (*:p<0.05) between two groups according to statistical analysis consistingof one-way ANOVA followed by pairwise comparisons and Tukey correction.

FIG. 35 is a graph showing interscapular brown adipose tissue (BAT)weight of mice treated by daily oral gavage with fresh and frozensuspensions of 5·10⁹ cells of D. welbionis J115^(T) and fed a HF-diet(HFD-J115-fresh and HFD-J115-frozen, respectively) or mice fed a controldiet (Control) or a high-fat diet (HFD) and treated by daily oral gavagewith an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group). Data are shown asscatter dot plot with median. Stars indicate significant differences (*:p<0.05) between two groups according to statistical analysis consistingof one-way ANOVA followed by pairwise comparisons and Tukey correction.

FIG. 36 is a graph showing body temperature of mice treated by dailyoral gavage with frozen suspensions of 5·10⁹ cells of D. welbionisJ115^(T) and fed a HF-diet (HFD-J115-fresh) or a high-fat diet (HFD) andtreated by daily oral gavage with an equivalent volume of steriletrehalose 15% in anaerobic PBS-carbonate buffer saline for 3 weeks(n=7/group). Data are shown as scatter dot plot with median. Starsindicate significant differences (**: p<0.01) between two groupsaccording to statistical analysis consisting of Mann-Whitney test.

FIG. 37 is a graph showing the correlation between BAT weight and bodytemperature of mice treated by daily oral gavage with frozen suspensionsof 5·10⁹ cells of D. welbionis J115^(T) and fed a HF-diet(H1-D-J115-fresh) or a high-fat diet (HFD) and treated by daily oralgavage with an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 3 weeks (n=7/group). Data are shown asscatter dot plot with median. The p value is indicated according tostatistical analysis consisting of Spearman correlation.

FIG. 38 is a graph obtained from RNAseq analysis in BAT, representingthe relative expression of genes belonging to inflammatory response geneontology group of mice treated by daily oral gavage with frozensuspensions of 5·10⁹ cells of D. welbionis J115^(T) and fed a HF-dietrelative to mice fed a high-fat diet (HFD) and treated by daily oralgavage with an equivalent volume of sterile trehalose 15% in anaerobicPBS-carbonate buffer saline for 13-weeks (n=12/group). Data are shown asthe ratio of expression in treated over expression in untreated mice.

FIG. 39 is a graph obtained from qPCR analysis in BAT, representing therelative expression of F4/80 in mice treated by daily oral gavage withfresh and frozen suspensions of 5·10⁹ cells of D. welbionis J115^(T) andfed a HF-diet (HFD-J115-fresh and HFD-J115-frozen, respectively) or micefed a control diet (Control) or a high-fat diet (HFD) and treated bydaily oral gavage with an equivalent volume of sterile trehalose 15% inanaerobic PBS-carbonate buffer saline for 13-weeks (n=12/group). Dataare shown as scatter dot plot with median. Stars indicate significantdifferences (*: p<0.05; **: p<0.01) between two groups according tostatistical analysis consisting of one-way ANOVA followed by pairwisecomparisons and Tukey correction.

FIG. 40 is a graph obtained from qPCR analysis in BAT, representing therelative expression of ColA1 in mice treated by daily oral gavage withfresh and frozen suspensions of 5·10⁹ cells of D. welbionis J1115^(T)and fed a HF-diet (HFD-J115-fresh and HFD-J115-frozen, respectively) ormice fed a control diet (Control) or a high-fat diet (HFD) and treatedby daily oral gavage with an equivalent volume of sterile trehalose 15%in anaerobic PBS-carbonate buffer saline for 13-weeks (n=12/group). Dataare shown as scatter dot plot with median. Stars indicate significantdifferences (**: p<0.01) between two groups according to statisticalanalysis consisting of one-way ANOVA followed by pairwise comparisonsand Tukey correction.

FIG. 41 is a graph showing the stimulation of human HEK-hTLR2 cells byincreasing concentration of frozen D. welbionis J115^(T).

FIG. 42 is a graph showing the effective concentration of frozen D.welbionis J115^(T) necessary to obtain half of the maximal stimulationof human HEK-hTLR2 cells (EC50).

FIG. 43 is a graph obtained from qPCR analysis in the jejunum,representing the relative expression of defensin alpha (DEFA) in micetreated by daily oral gavage with fresh and frozen suspensions of 5·10⁹cells of D. welbionis J115^(T) and fed a HF-diet (HFD-J115-fresh andHFD-J115-frozen, respectively) or mice fed a control diet (Control) or ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n=12/group). Data are shown as scatter dot plot withmedian. Stars indicate significant differences (*: p<0.05; **: p<0.01)between two groups according to statistical analysis consisting ofone-way ANOVA followed by pairwise comparisons and Tukey correction.

FIG. 44 is a graph showing that faecal Dysosmobacter spp relativeabundance in correlates negatively with body mass index (BMI) in humans.Pearson correlation between the BMI and the log of Dysosmobacter spprelative abundance in stools.

EXAMPLES

The present invention is further illustrated by the following examples.

Example 1: Isolation of Strain J115 from a Faecal Sample of a Healthy 25Years Old Female

For determining that an isolate is a new species, several analysesshould be performed: morphological (such as motility and flagella),biochemical (such as enzymatic and fermentation capacity), physiological(such as fatty acid analysis) and phylogenetic (such as Averagenucleotide identity (ANI), DNA-DNA hybridization and GC content)characterization.

Fecal sample was kept in a sealed container with an O₂-absorbing andCO₂-generating agent (Genbox Anaer; Biomérieux) and isolation wasperformed less than two hours after collection. The sample wastransferred into an anaerobic chamber (Coy) containing 100% N₂ as gasatmosphere and immediately diluted 1/10 in modified YCFA (Yeastextract—casein hydrolysate—fatty acids) enriched in antioxidants (Table1 and Table 2).

TABLE 1 Composition of the modified YCFA medium. Quantity for 1 LIngredient of medium Yeast extract 8 g Soy peptone 4 g Wheat peptone 4 gKH2PO4 5 g Na₂CO₃ 4 g NaCl 1 g MgCl₂ 50 mg MgSO₄ 50 mg CaCl₂ 50 mg Hemin10 mg Resazurin solution (1 g/L) 1 mL Glucose 10 g Cystein 1 gGlutathion reduced 1 g Ascorbate 0.5 g Uric acid 0.3 g Vitamin solution1 mL H₂O q.s. 1000 mL q.s.: quantum satis.

TABLE 2 Composition of the vitamin solution used to prepare the modifiedYCFA medium. Quantity for 1 L Vitamins solution of solution (mg) Biotin2 Folic acid 2 Pyridoxine-HCl 10 Thiamine-HCl × 2 H 2 Riboflavin 5Nicotinic acid 5 D-Ca-pantothenate 5 Vitamin B 12 p-Aminobenzoic acid 5Lipoic acid 5 H₂O q.s. 1000 q.s.: quantum satis.

Fecal suspension was then transferred in tubes hermetically sealed withbutyl rubber under an atmosphere of 20% CO₂ —80% N₂. Then, single-cellcultivation was performed using extinction dilution technique, i.e., thefecal suspension was diluted and aliquoted in 300 vials such that asingle vial received on average one cell. Positive cultures after 24 hto 7 days at 37° C. were spread onto solid modified YCFA and incubated72 h to 7 days at the same temperature in anaerobic jars (Merck) with anO₂-absorbing and CO₂-generating agent (Genbox Anaer; Biomérieux). Singlecolonies were picked and transferred to fresh medium and the process wasrepeated until the cultures were deemed pure. Among the culturesobtained, one, designated J115, was considered for further study. Fortyheight hours cultures in modified YCFA medium were used for routineincubation, growth tests and biochemical analyses. The strain was storedat −80° C. in 20% glycerol.

The J115 strain was identified as related to species of the genusOscillibacter, O. valericigenes and O. ruminantium (see sectionphylogenetic analysis hereinafter). Therefore, the type strains for eachof these 2 species was obtained for comparison purpose. O. ruminantiumJCM 18333^(T) (=GH1T=KCTC 15176=NBRC 108824) was obtained from the JapanCollection of Microorganisms (JCM) while O. valericigenes DSM 18026^(T)(Sjm18-20T=NBRC 101213) was obtained from the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ). Forty-eight hours cultures inmodified YCFA medium at 37° C. were used for routine incubation, growthtests and biochemical analyses. The strains were stored at −80° C. in20% glycerol.

Scanning electron microscopy showed that J115 cells were straight rods,occurred singly and measured mainly 0.5-0.6×1.8-3.0 μm but rods up to18-20 μm long were regularly observed during exponential and earlystationary phase (FIG. 1). J115 colonies on solid modified YCFA after 72h of incubation at 37° C. in anaerobic atmosphere were punctiform,cream, translucent, circular, entire, slightly convex and smooth. Strainwas negative for motility when stab-inoculated into semi-solid modifiedYCFA (0.5% agar) and anaerobically incubated at 37° C. for 72 h.Contrary to J115 cells, O. valericigenes DSM 18026^(T) and O.ruminantium JCM 18333^(T) cells are motile and have a flagellum.

The ability to tolerate bile and NaCl was tested in liquid modified YCFAcontaining increasing concentration of bovine bile (Sigma, 1% w/v ofdehydrated bile corresponding to 10% w/v fresh bile) or NaCl (VWR).Growth of the strain occurred on medium containing below 2% bile or 2%NaCl but not on medium containing 2.5% or on medium containing 2.25% orabove bile or NaCl. O. ruminantium JCM 18333^(T) could grow on mediumhaving 0 to 2% bile while O. valericigenes DSM 18026^(T) could grow inmedium containing 0 to 1% bile.

Gram staining was negative. KOH test (3%, w/v) was positive. No sporeformation was observed in transmission or scanning electron microscopy(Philips Electron Microscope CM12/STEM) at exponential and stationarygrowing phase. No growth occurred after a 30 min treatment with 70°ethanol. No catalase was detected using 3% w/v H₂O₂ test for isolateJ115 and the strain grew only in strict anaerobic conditions.

Biochemical Characterization

The results of biochemical characterization are given in the speciesdescription, Table 3 and Table 4. The rapid ID 32A anaerobeidentification kit (Biomérieux) was used according to the manufacturerinstruction and the API 20A anaerobe test kit and the API 50CHcarbohydrates kit (Biomérieux) with modified YCFA without carbon source.Tests were performed in triplicate on three separate cultures. Rapid ID32A and API 20A showed that strain J115 was positive for glutamic aciddecarboxylase and arginine dihydrolase but negative for all the othertests (Table 3).

TABLE 3 biochemical characterization of strain J115, O. valericigenesDSM 18026^(T) and O. Ruminantium JCM 18333^(T). Results from Rapid ID32Aand API 20A tests are indicated as follow “−” indicates a negative test,“+” indicates a positive test. Enzymatic activity J115 DSM 18026^(T) JCM18333^(T) Indole production − − − Nitrates reduction − − − Gelatinase −− − Esculinase − − + Urease − − − Alkaline phosphatase − − − Argininedihydrolase + + + α-galactosidase − − − β-galactosidase − − −β-galactosidase-6-phosphate − − − α-glucosidase − − − β-glucosidase − −− α-arabinosidase − − − β-glucuronidase − − − N-acetyl-β-glucosaminidase− − − α-fucosidase − − − Glutamic acid decarboxylase + + + Argininearylamidase − − − Proline arylamidase − − − Leucyl glycine arylamidase −− − Phenylalanine arylamidase − − − Leucine arylamidase − − −Pyroglutamic acid arylamidase − − − Tyrosine arylamidase − − − Alaninearylamidase − − − Glycine arylamidase − − − Histidine arylamidase − − −Glutamyl glutamic acid arylamidase − − − Serine arylamidase − − −

Acid production by fermentation from various carbon sources was testedusing API 50CH kit and is was found that strain J115 could bedifferentiated from both Oscillibacter species by its ability to fermentmyo-inositol and its inability to ferment D-glucose, and D-xylose (Table4).

TABLE 4 fermentation capacity of strain J115. na: not available. Resultsfrom the API 50 CH test are presented as follow: “−” indicates asubstrate not fermented by the strain considered and “+” indicates asubstrate fermented by the strain considered O. O. valerecigenesruminantium J115 DSM 18026^(T) JCM 18333^(T) Glycerol − − − Erythritol −− − D-arabinose − − − L-arabinose − − + D-ribose − − + D-xylose − + +L-xylose − − − D-adonytol − − − Methyl-βD-xylopyranoside − − −D-galactose − − − D-glucose − + + D-fructose − − − D-mannose − − −L-sorbose − − − L-rhamnose − − − Dulcitol − − − Myo-inositol + − −D-mannitol − − − D-sorbitol − − − Methyl-αD-mannopyranoside − − −Methyl-αD-glucopyranoside − − − N-acethyl-glucosamine − − − Amygdaline −− − Arbutine − − − Salicin − − − D-cellobiose − − − D-maltose − − −D-lactose − − − D-melibiose − − − Sucrose − − − D-trehalose − − − Inulin− − − D-melezitol − − + D-raffinose − − − Starch − − − Glycogen − − −Xylitol − − − Gentibiose − − − D-turanose − − − D-lyxose − − − L-lyxose− − − D-tagatose − + − D-fucose − − − L-fucose − − − D-arabitol − − −L-arabitol − − − Potassium gluconate − − − Potassium 2-cetogluconate − −− Potassium 5-cetogluconate − − −

Cellular Fatty Acid Composition

Cellular fatty acids were analyzed by the Identification Service of theDSMZ, Braunschweig, Germany from 30 mg of freeze-dried cells bysaponification, methylation and extraction using minor modifications ofthe method of Miller (J Clin Microbiol. 1982 September; 16(3):584-6) andKuykendall et al. (Int J Syst Evol Microbiol. 1988; 38(4):358-361). AsO. valericigenes Sjm18-20T and O. ruminantium GH1T, the major cellularsaturated branched-chain fatty acids of strain J115 are iso-C15:0(24.2%) and anteiso-C15:0 (15.2%) (Table 5). However, the quantitiesdiffered quite substantially as iso-C_(15:0) represented only 9.8 and8.9% of the cellular fatty acids in the Oscillibacter species.Furthermore, anteiso-C_(15:0) are not major cellular fatty acids of O.valericigenes DSM 18026^(T) and O. ruminantium JCM 18333^(T).Conversely, the saturated straight fatty acids C14:0 and C16:0 are majorcellular fatty acids of O. valericigenes DSM 18026^(T) and O.ruminantium JCM 18333^(T) but are detected only in trace amount instrain J115.

TABLE 5 Cellular fatty acids analysis. Strains: 1, J115; 2, O.valericigenes Sjm 18-20T; 3, O. ruminantium GH1T. Fatty acid 1 2 3Saturated straight-chain C_(12:0) Tr 1.2 5.3 C_(14:0) 2.4 14.7  11.5 C_(15:0) Tr 1.7 Tr C_(16:0) Tr 8.7 14.3  C_(18:0) Tr Tr 1.7 Unsaturatedstraight-chain C18:2ω6,9c Tr Tr Tr Dimethylacetal (DMA) C_(14:0) DMA Tr6.5 4.5 anteiso-C_(15:0) DMA 1.0 Tr Tr C_(16:0) DMA 7.6 25.2  19.9 C_(17:0) DMA 2.2 Tr Tr C_(18:0) DMA 18.4  — — Aldehydes C_(16:0) ALDE1.1 3.8 4.3 C_(18:0) ALDE 4.3 — — Saturated branched-chain iso-C_(13:0)Tr 1.3 11.8  iso-C_(14:0) 1.5 Tr Tr iso-C_(15:0) 24.2  9.8 8.3iso-C_(16:0) Tr — — anteiso-C_(13:0) Tr Tr 1.2 anteiso-C_(15:0) 15.2 3.0 3.0 iso-C_(17:0) Tr — — anteiso-C_(17:0) Tr — — Summed features* 1Tr 1.0 Tr 3 Tr Tr Tr 5 Tr Tr 1.9 Tr, trace amount (<1%); —, notdetected. *Summed features represent groups of two or three fatty acidsthat could not be separated using the MIDI Sherlock system. Summedfeature 1 contains C_(13:1)ω1c and/or C_(14:0) ALDE. Summed feature 3contains one or more of an unknown fatty acid of ECL 13.570 and/oriso-C_(15:0) ALDE. Summed feature 5 contains C_(15:0) DMA and/orC_(14:0) 3-OH.

However, strain J115 differs from those two species by the fattyaldehydes found as dimethylacetals (DMAs) such as C_(18:0) DMA which isabundant in J115 (18.4%) but absent from Oscillibacter species.Moreover, C_(16:0) DMA are found in much lower concentrations in strainJ115 than in Oscillibacter species. C_(16:0) DMA represented 25.2 and19.9% of the cellular fatty acids in the two Oscillibacter species. Inaddition, C_(14:0) DMA is detected in appreciable amount in O.valericigenes DSM 18026^(T) and O. ruminantium JCM 18333^(T) (6.2 and4.5%, respectively) but represents only trace amount of the cellularfatty acids of strain J115.

Respiratory Lipoquinones

Respiratory lipoquinones and diaminopimelic acid of strain J115 wereanalyzed by the Identification Service of the DSMZ, Braunschweig,Germany. Briefly, quinones were extracted from 100 mg of freeze driedcells using methanol:hexane, followed by phase separation into hexaneaccording to Tindall's method. As in Oscillibacter species, no quinonewas detected in strain J115. Whole cell hydrolysates were examined bythin layer chromatography on cellulose plates using the solvent systemof Rhuland et al. (J Am Chem Soc 1955; 77:4844-6). Strain J115 containedmeso-2,6-diaminopimelic acid as the diagnostic diamino acid of thecell-wall peptidoglycan.

Analysis of DNA base composition was carried out by the IdentificationService and Dr Peter Schumann, DSMZ, Braunschweig, Germany. The DNA GCcontent of strain J115 was 59.3%, slightly higher of those of those ofO. valericigenes Sjm18-20T and O. ruminantium GH1T (52.9% and 54.9%respectively).

Phylogenic Analyses

An almost complete (1428 bp—SEQ ID NO: 1) 16S rRNA sequence of strainJ115 was obtained using the universal primers 8F(5′-AGAGTTTGATCCTGGCTCAG-3′ SEQ ID NO: 2) and 1492R(5′-GGTTACCTTGTTACGACTT-3′ SEQ ID NO: 3). 16S rRNA sequences of theclosest previously identified relatives of strain J115 were determinedand retrieved using EzBioCloud's Identify service (database updated 2017Oct. 23) and GenBank Database®.

Multiple alignment of the sequences was performed using MUSCLE.Distances were computed using the Maximum Composite Likelihood methodand the phylogenetic tree was constructed by the neighbor-joining methodin MEGA 7.0 after gaps and unknown bases were eliminated. Strain J115falls within cluster IV of the low GC content clostridial bacteriabranch. Strain J115 was related to Oscillibacter ruminantium GH1T (95.4%similarity) and Oscillibacter valericigenes Sjm 18-20T (94.1%similarity). Strain J115 was also located near the Oscillospiraguilliermondii clade. Phylogenetically, strain J115^(T) formed amonophyletic separate branch that was located as a sister clade to theOscillibacter-Oscillospira clade supported by a 100% bootstrap value inboth neighbor-joining and maximum likelihood trees (FIG. 2 A and B).

For whole genome sequence, high molecular weight DNA was extracted usingQiagen DNeasy UltraClean Microbial kit. Long-reads were obtained usingPacBio technology at Eurofins GATC company. Assembly was performed usinghierarchical genome-assembly process and produced a 3 576 111 base pairscomplete genome in one contig [NCBI accession number CP034413], whosedepth of coverage was 242. Genome analysis using ContEst16S algorithmindicated that the genome of strain J115^(T) was not contaminated. Thesequences of three 16S rRNA gene copies were retrieved and compared tothe 16S rRNA gene sequence obtained by PCR and Sanger sequencing. Thethree copies were strictly identical to the sequence obtained by PCR.

Multilocus sequence analysis (MLSA) was performed to obtain a higherresolution of the phylogenetic relationships between strain J115 andneighbour taxa belonging to the Ruminococcaceae family. The sequences of12 protein-coding genes (sigE, purM, ass, lysC, phoH, crh, groEL, thdF,infB, recA, rpoD and gyrB) were retrieved from the complete genomes ofall type strains except from Oscillispira guillermondii and Flintibacterbutyricus whose complete genomes are not available. Similarly, to the16S rRNA gene analysis, the concatenated sequences were aligned usingMUSCLE and phylogenetic trees were constructed by the neighbor-joiningand maximum likelihood methods with 1000 bootstraps replications (FIG. 3A and B). As previously, J115 formed a monophyletic separate branch thatwas located as a sister clade to the Oscillibacter clade supported by a100% bootstrap value in both trees.

A robust measurement of genomic relatedness between strain, the Averagenucleotide identity (ANI), strain J115 and available reference strainsgenomes were calculated using OAT standalone 0.93.1 software. The ANIscores were represented as a heatmap and used to construct a dendrogramwith unweighted pair group method with arithmetic mean (UPGMA) (FIG. 4).An ANI score obtained with reference species or strain that is higherthan 98.65% (or 98.7%) implies that the isolate is a new species. As in16S rRNA gene sequence and MLSA, the closest taxa to strain J115^(T)were O. ruminantium JCM 18333^(T) and O. valericigenes DSM 18026^(T)with ANI scores of 73.37 and 73.24, respectively.

To complement this analysis based on ANI, intergenome distance wascalculated using the genome to genome calculator 2.1 provided by DSMZ.The settings used were BLAST+ as local alignment tool and formula 2,that is to say the sum of all identities found in high-scoring segmentpairs (HSP) divided by overall HSP length. Intergenome distances werethen used to determine the probability to have a DNA-DNA hybridization(DDH) equal or above 70% and to generate a heatmap along with a UPGMAtree using OAT standalone 0.93.1 software (FIG. 5). The UPGMA tree basedon intergenome distances had a quite different topology from thepreviously obtained trees. Indeed, the closest species with standing inNomenclature according to intergenome distance was Faecalibacteriumprausnitzii ATCC 27768^(T) with an intergenome distance of 0.1342 whichcorresponds to a probability of DDH equal or above 70% of 0.19 and an insilico DDH value of 31.5 [29.1-34]%. Despite the divergences to the 16SrRNA gene sequence, MLSA and ANI based results, in silico DDH resultsclearly indicate that strain J115 belongs to the Ruminococcaceae familyin Clostridial cluster IV and differs significantly from the closesttaxon with standing in Nomenclature: the Oscillibacter genus.

Conclusion

Table 6 summarizes the differences observed between strain J115 and thetype strains of the closest related species Oscillibacter. ruminantiumJCM 18333^(T) (=GH1T=KCTC 15176=NBRC 108824) Oscillibacter valericigenesDSM 18026^(T) (Sjm18-20T=NBRC 101213).

TABLE 6 Characteristics of strain J115 (Dysosmobacter welbionis), O.valericigenes DSM 18026^(T) and O. ruminantium JCM 18333^(T)Characteristic 1 2 3 Source of isolation Human Alimentary canal rumen ofgut of Japanese Korean corbicula

native cattle Motility Non-motile Motile Motile Flagella Absent PresentPresent GC content (mol %) 59.3 52.7 54.6 by HPLC GC content (mol %)58.9 53.2 55.0 based on genome data Growth bile   0-2%   0-1%   0-2%concentration Growth NaCl 0-1.4% 0-3.5% 0-2.5% concentrationFermentation capacity myo-inositol + − − D-xylose − + + D-arabinose −− + D-ribose − − + D-glucose − + + D-melezitol − − + Tagatose − + −Enzymatic activity Aesculinase + − + Sequence-based analyses 16S mRNAidentity with 94.1% 95.4% J115 Average Nucleotide 73.24 73.37 Identity(ANI) with J115 Intergenome distance 0.22 0.24 with

indicates data missing or illegible when filed

Strain J115 16S rRNA gene sequence diverges from those of O. ruminantiumGH1T and O. valericigenes Sjm 18-20T by 4.6-5.9%, which is lower thanthe proposed threshold of 6% for prokaryotic genus delineation. Cells ofstrain J115 were straight rods, normally 1.8-3.0 μm and often formelongated rods. Strain J115 was strictly anaerobic and had norespiratory quinone. These properties are similar to those ofOscillibacter species. However, strain J115 was non-motile, had noflagella and had different cellular fatty acids composition. Inaddition, strain J115 was not able to utilize glucose, and xylose on thecontrary to species belonging to Oscillibacter genus but was able toferment myo-inositol. Phylogenetically, strain J115 formed a separatebranch to the clade Oscillibacter-Oscillospira. These two subclades arealready accommodated as two separate genera. On the basis of itsphylogenetic position and biochemical and physiological propertiesdescribed above, strain J115 differs significantly from the nearestcultivated genus members, namely Oscillibacter ruminantium andOscillibacter valericigenes. Consequently, strain J115 represents anovel species of a new genus, for which the name Dysosmobacter welbionisgen. nov. sp. nov. is proposed.

Example 2: Description of Dysosmobacter Gen. Nov

Dysosmobacter cells are obligatory anaerobic, non-pigmented,non-spore-forming, non-motile, Gram-stain-negative. Cells form straightrods mainly 1.8-3.0 μm but often form elongated rods whatever thegrowing phase. No respiratory menaquinones are produced. The diagnosticdiamino acid in the cell wall is meso-2,6-diaminopimelic acid. The genusis a member of the family Ruminococcaceae. The type species isDysosmobacter welbionis.

Example 3: Description of Dysosmobacter Welbionis Sp. Nov

Dysosmobacter welbionis exhibits the following characteristics inaddition to those in the genus description. Colonies on solid modifiedYCFA after 72 h of incubation at 37° C. under anaerobic conditions arepunctiform, cream, translucent, circular, entire, slightly convex andsmooth. Growth is inhibited by the presence of 2% bile or 2% NaCl.Aesculin is not hydrolysed. Indole is not produced. Nitrate is notreduced. Gelatin is not digested. Urease is not produced. Catalase isnot produced. Acid is produced from myo-inositol but not from D-glucose,D-arabinose, D-ribose and D-xylose. Positive reactions are obtained forarginine dihydrolase and glutamic acid decarboxylase. All the othertests from API 20A and Rapid ID 32A are negative (i.e. alkalinephosphatase, α-galactosidase, β-galactosidase, α-glucosidase,β-glucosidase, α-arabinosidase, β-glucuronidase,N-acetylglucosaminidase, α-fucosidase, arginine arylamidase, prolinearylamidase, leucyl-glycine arylamidase, phenylalanine arylamidase,pyroglutamic acid arylamidase, tyrosine arylamidase, alaninearylamidase, glycine arylamydase, histidine arylamidase, serinearylamidase). Acid is produced from myo-inositol but not fromD-adonytol, amygdaline, D/L-arabinose, D/L-arabitol, arbutine,D-cellobiose, dulcitol, erythrol, D-fructose, D/L-fucose, D-galactose,gentibiose, D-glucose, glycerol, glycogen, inulin, D-lactose, lyxose,D-maltose, D-mannitol, D-mannose, D-melezitol, D-mellibiose,methyl-αD-glucopyranoside, methyl-αD-mannopyranoside,methyl-βD-xylanopyranoside, N-acethylglucosamine, D-raffinose,L-rhamnose, D-ribose, D-saccharose, salicin, D-sorbitol, L-sorbose,starch, tagatose, D-trehalose, D-turanose, xylitol and D/L-xylose).Major fermentation end-products from myo-inositol are butyrate (orbutyric acid). The DNA GC content of the type strain is 58.92 mol %.Major cellular fatty acids are saturated branched-chain fatty acids andDMAs. Major DMA fatty acid is C_(18:0) DMA and major saturatedbranched-chain fatty acids are iso-C_(15:0) and anteiso-C_(15:0). Thespecies type strain is, J115 (deposited at the BCCM/LMG on Mar. 14, 2018as LMG P-30603) was isolated from human feces. Strain J115 is hence alsoreferred to herein as strain J115^(T).

Example 4: Modification to the Description of Related Taxa

Observations made during the comparison of the characteristics of J115with the characteristics of the type strains of the related species,Oscillibacter valericigenes and Oscillibacter ruminantium lead to anecessary update to the previously published description of the relatedgenera Oscillibacter and of the related species Oscillibactervalericigenes and Oscillibacter ruminantium.

Emended Description of the Genus Oscillibacter

The description is as given by Iino et al. (Int J Syst Evol Microbiol2007; 57:1840-5) with the following modifications. Positive for glutamicacid decarboxylase and arginine dihydrolase. Negative for alkalinephosphatase, α-galactosidase, β-galactosidase, α-glucosidase,β-glucosidase, α-arabinosidase, β-glucuronidase,N-acetylglucosaminidase, α-fucosidase, arginine arylamidase, prolinearylamidase, leucyl-glycine arylamidase, phenylalanine arylamidase,pyroglutamic acid arylamidase, tyrosine arylamidase, alaninearylamidase, glycine arylamydase, histidine arylamidase, serinearylamidase. Indole is not produced from tryptophane. Gelatin is notdigested.

Emended Description of Oscillibacter valericigenes

The description is as given by Iino et al. (Int J Syst Evol Microbiol2007; 57:1840-5) with the following modifications. Aesculin is nothydrolysed. The bile concentration range allowing growth is 0-1%. Acidis produced from tagatose. Acid is not produced from D/L-arabinose andD-ribose.

Emended Description of Oscillibacter ruminantium

The description is as given by Lee et al. (Int J Syst Evol Microbiol2013; 63:1942-6) with the following modifications. Aesculin ishydrolysed. The bile concentration range allowing growth is 0-2%. Acidis produced from D-arabinose and D-melezitol.

Example 5: Effects of the Administration of Dysosmobacter Welbionis onMetabolism, Feeding Behaviour and Intestinal Barrier Material andMethods Mice

A set of 10-week-old C57BL/6J mice (30 mice, n=10/group) (Janvier Labs,France) were housed in groups of 2 mice/cage, with free access to foodand water. The mice were fed a control diet (CT) (AIN93Mi; Researchdiet, New Brunswick, N.J., USA) or a high-fat diet (HFD) (60% fat and20% carbohydrates (kcal/100 g), D12492, Research diet, New Brunswick,N.J., USA). Mice were treated with an oral administration ofDysosmobacter welbionis by oral gavage at the dose 1·10⁹ cfu/0.2 mLsuspended in sterile anaerobic phosphate-carbonate buffer saline(HFD-Dysosmobacter J115) and control groups were treated with an oralgavage of an equivalent volume of sterile anaerobic phosphate-carbonatebuffer saline (Control and HFD). Treatment continued for 8 weeks.

D. welbionis J115 (deposited at the BCCM/LMG on Mar. 14, 2018 as LMGP-30603) was grown anaerobically in a modified YCFA medium enriched ininositol as described in Example 1 and then washed and suspended inanaerobic phosphate-carbonate buffer saline to an end concentration of1·10⁹ cfu/0.2 mL.

Food and water intake were recorded once a week. Body composition wasassessed by using 7.5 MHz time domain-nuclear magnetic resonance(TD-NMR) (LF50 minispec, Bruker, Rheinstetten, Germany).

All mouse experiments were approved by and performed in accordance withthe guidelines of the local ethics committee. Housing conditions werespecified by the Belgian Law of Apr. 6, 2010, regarding the protectionof laboratory animals (agreement number LA1230314).

Dysosmobacter Welbionis J115^(T) Cultivation and Enumeration

D. welbionis J115^(T) (LMG P-30603) was grown anaerobically in amodified YCFA medium enriched with inositol as previously described (LeRoy et al, published in Int J Syst Evol Microbiol, 2019, DOI10.1099/ijsem.0.003547).

Tissue Sampling

The animals were anesthetized with isoflurane (Forene®, Abbott,Queenborough, Kent, England) before exsanguination and tissue sampling,then mice were killed by cervical dislocation. The intestinal segments(duodenum, jejunum, ileum, cecum and colon) were dissected, immersed inliquid nitrogen, and stored at −80° C., for further analysis.

RNA Preparation and Real-Time qPCR Analysis

Total RNA was prepared from tissues using TriPure reagent (Roche).Quantification and integrity analysis of total RNA was performed byrunning 1 μL of each sample on an Agilent 2100 Bioanalyzer (Agilent RNA6000 Nano Kit, Agilent).

cDNA was prepared by reverse transcription of 1 μg total RNA using aReverse Transcription System kit (Promega, Leiden, The Netherlands).Real-time PCRs were performed with the StepOnePlus™ real-time PCR systemand software (Applied Biosystems, Den Ijssel, The Netherlands) usingMesa Fast gPCR™ (Eurogentec, Seraing, Belgium) for detection accordingto the manufacturer's instructions. RPL19 was chosen as the housekeepinggene. All samples were run in duplicate in a single 96-well reactionplate, and data were analyzed according to the 2^(−ΔΔCt) method. Theidentity and purity of the amplified product was checked throughanalysis of the melting curve carried out at the end of amplification.Primer sequences for the targeted mouse genes are presented in the Table7 below.

TABLE 7 Nucleotide sequence of the primer pairs used forthe measurement of mRNAs expression level by qPCR. SEQ ID PrimersSequence NO: RPL-19 Forward GAAGGTCAAAGGGAATGTGTTCA 4 ReverseCCTGTTGCTCACTTGT 5 Proglucagon Forward TGGCAGCACGCCCTTC 6 ReverseGCGCTTCTGTCTGGGA 7 Occludin Forward ATGTCCGGCCGATGCTCTC 8 ReverseTTTGGCTGCTCTTGGGTCTGTAT 9

Intestinal Energy Absorption

Six weeks after the beginning of the experiment, the feces of twoseven-days periods were collected. During the same time the food intakewas monitored. The feces were dried at 60° C. during 2 hours andweighted. Total energy of the diet and the feces was determined by bombcalorimetry (C1, IKA, USA). The net intestinal absorption is calculatedbased on the ingested and excreted energy and represented the proportionof ingested energy that was not recovered in feces output.

Statistical Analysis

Data are expressed as means±s.e.m. Differences between groups wereassessed by one-way ANOVA followed by pairwise comparisons and testedfor false discovery rate using the two-stage step-up method ofBenjamini, Krieger and Yekutieli. Correlations were assessed by Pearsonor non-parametric Spearman tests depending on if the variables passedShapiro-Wilk normality test or not. Data were analyzed using GraphPadPrism version 7.00 for windows (GraphPad Software, San Diego, Calif.,USA). Results were considered statistically significant when p<0.05.

Results

To decipher if and how Dysosmobacter welbionis affects host metabolichealth, the bacterium J115 was orally administered to high-fat fed miceduring eight weeks. Results show that supplementation with 10⁹ D.welbionis cells per day decreased by 29% (i.e. 1.5 grams) high-fatinduced weight gain (FIG. 6 and FIG. 7). Additionally, results also showthat D. welbionis administration significantly decreased food intake andthus total energy intake by 5% over the course of the experiment (FIG. 8and FIG. 9). To verify that this decreased food intake indeed resultedin a decrease in the energy available for host metabolism, the feces oftwo seven days periods were collected and the total energy contained inthose feces was measured by bomb calorimetry. Energy output in the feceswas decreased by D. welbionis administration (FIG. 10), however, the netintestinal absorption remained 7.7% lower than in HFD mice (FIG. 11).Furthermore, the ratio of weight gain over food intake was 15.5% lowerin D. welbionis supplemented mice than in HFD mice (FIG. 12). Thisindicates that D. welbionis decreased the ability to convert food energyinto body mass. Taken together, these results show that D. welbionisprevents high fat diet induced weight gain by two means: a decrease offood intake and a decrease of the ability to store the absorbed energy.

Glucagon-like peptide-1 (GLP1) is a protein secreted by the intestine inthe blood that regulates satiety and thus, food intake, both in humanand animals GLP1 in encoded by the proglucagon gene, which is expressedby L-cells of the intestine mucosa. The expression of the proglucagongene in the ileum was measured and it was found that D. welbionisadministration increased proglucagon ileal expression by 26% (FIG. 13).

As short chain fatty acids (SCFA) production by intestinal microbiotahas a regulatory role on host metabolism and immunity, both locally andperipherally, SCFA production by D. welbionis was measured in vitro.SCFA are end-products of carbohydrates fermentation by intestinalbacterial, they are known to activate G protein-coupled receptors (GPCR)41 and 43, which are found at the apical membrane of L-cells. GPCR41/43activation leads to a strengthening of the gut barrier through astimulation of the expression of tight junction proteins. Tight junctionproteins are proteins found at the lateral membrane of epithelial cellsin the intestine and their junction ensures the integrity of theepithelium. D. welbionis produced large amounts of SCFA, butyrate inparticular (FIG. 14). The inventors thus hypothesized that D. welbionisadministration strengthens gut barrier and measured the mRNA expressionof the gene coding for the tight junction protein Occludin and Claudin 3(FIG. 15 and data not shown). Consistently with this hypothesis, anincrease in the expression of occludin and claudin 3 was found in theileum of mice treated with D. welbionis.

Conclusion

Together, these observations all point to a role of D. welbionis in themetabolism and integrity of the intestinal epithelial barrier in thehost organism.

More specifically, the daily administration of 10⁹ J115 cells in mice,lead to the amelioration of several deleterious consequences associatedwith an high fat diet: a decrease of the gain of weight, a decrease infood intake, a decrease of the gain of weight per food intake, adecrease in energy absorption in the intestine and the increase ofproglucagon mRNA expression. These observations point to the beneficialeffect of the administration of J115 for the treatment of metabolicdiseases (notably obesity) and feeding behavior disorders.

In addition, the daily administration of 10⁹ J115 cells in mice increasethe amount of SCFA and the expression of tight junction markers,suggesting a reinforcement of the intestinal barrier associated with theadministration of J115. These observations point to a beneficial effectof the administration of J115 for the treatment of diseases associatedwith a dysfunctional intestinal barrier notably intestinalinflammations, Crohn's disease, ulcerative colitis, food allergies,celiac disease, ulcers, infection, non-alcoholic steatohepatitis, coloncancer.

Example 6: Effects of the Administration of D. Welbionis on GLP-1Production and Secretion Material and Methods In Vitro GLP-1 ProductionAssay

Enteroendocrine cells from the intestinal murine L cell line GLUTagcells were used from passage 17 to 28. Cells were grown in DMEM GlutaMAXsupplemented with 10% (v/v) inactivated FBS and 1% (v/v)penicillin/streptomycin, at 37° C. in a 5% CO₂/95% air atmosphere.GLUTag cells (1.8×10⁵ cells/well) were seeded into 24-well cell cultureplates, 500 μL per well, and allowed to adhere for 24 h. The day after,cells were treated for 2 h with D. welbionis J115^(T) at concentrationsranging from 1·10⁷ to 2·10⁹ cells/mL in the presence of DPP-IV inhibitor(dipeptidyl peptidase-4) at 50 μM final concentration. Total GLP-1(glucagon like peptide-1) concentrations were determined with the MesoScale Discovery ELISA kits (MesoScale, Gaithersburg, USA) and expressedas the amount of GLP-1 detected in the supernatant, and the total amountof GLP-1 in the medium plus cells.

Results

As GLP-1 secretion by enteroendocrine cells not only contributes tostrengthen the intestinal barrier but also reduce food intake, we soughtto confirm that D. welbionis J115^(T) triggers GLP-1 production andsecretion by enteroendocrine cells. Thus, cells from the intestinalmurine L cells line GLUTag were subjected to increasing concentration ofD. welbionis J115^(T). The extracellular concentration of total GLP-1was not increased by 1·10⁷ cells/mL but was increased by 16.2%, 79.5%,119.69% and 199.7% when exposed to increased concentrations of D.welbionis J115^(T) such as 1·10⁸, 5·10⁸, 1·10⁹ and 2·10⁹ cells per mL,respectively (FIG. 16). This result demonstrates that D. welbionisJ115^(T) induces GLP-1 secretion by enteroendocrine cells. In order todecipher if D. welbionis J115^(T) also raises the production of GLP-1,the total quantity of GLP-1 produced by the GLUtag cells during thechallenge was measured, that is to say the sum of intracellular andextracellular amounts of GLP-1. The total amount of GLP-1 was notaffected by 1·10⁷ cells/mL but was increased by 32.9%, 19.5%, 38.5% and54.3% by concentrations of 1·10⁸, 5·10⁸, 1·10⁹ and 2·10⁹ cells per mL,respectively (FIG. 17). Therefore, showing that D. welbionis J115^(T)increases endogenous production of glucagon-like peptides (e.g., GLP1and GLP-2).

Conclusion

Consistently with the increase expression of proglucagon observed inmice terminal ileum following D. welbionis J115^(T) administration, adose-dependent increase of both secreted and total GLP-1 followingexposure to D. welbionis J115^(T) is observed. As GLP-2 production isassociated with the increase of GLP-1 production (Drucker et al., BestPract Res Clin Endocrinol Metab. 2004 December; 18(4):531-54) Theseresults are indicative of a role stimulatory effect of D. welbionisJ115^(T) on GLP-1 and GLP-2 production and secretion.

GLP-1 is associated with the reduction of energy intake, higher energyexpenditure, higher secretion of insulin, lower insulin resistance andsatiety. GLP-2 is associated with a strengthening of the gut barrier, aninduction of proliferative and cytoprotective pathways in the smallbowel the intestinal barrier Therefore, these observations also point toa beneficial effect of the administration of J115 for the treatment ofdiseases associated with a dysfunctional intestinal barrier and/orfeeding disorders.

Example 7: Influence of Dose and Viability on the Effect Associated withthe Administration of D. Welbionis Material and Methods DysosmobacterWelbionis J115^(T) Cultivation and Enumeration

D. welbionis J115^(T) (LMG P-30603) was grown anaerobically in amodified YCFA medium enriched with inositol as previously described (LeRoy et al, published in Int J Syst Evol Microbiol, 2019, DOI10.1099/ijsem.0.003547).

Fresh cultures of D. welbionis J115^(T) were prepared each dayadministered to the mice belonging to the HFD-J115 fresh group. Cultureswere transferred into 50 mL tubes in an anaerobic chamber, thencentrifuged at 5000 g during 20 min. Then, the supernatant was removedand the pellet resuspended in the appropriate volume of or trehalose 15%in phosphate-carbonate buffer saline to obtain the desired endconcentration in number of cells/mL, calculated from the culture'soptical density.

Frozen suspensions of D. welbionis J115^(T) were prepared in one batchbefore the beginning of the experiment and then frozen, an aliquot wasthawed every day for the daily administration to mice and was depictedas the HFD J115 frozen group. Briefly, cultures were transferred into 50mL tubes in an anaerobic chamber, then centrifuged at 5000 g during 20min. Then, the supernatant was removed and the pellet resuspended in theappropriate volume of trehalose 15% in phosphate-carbonate buffer salineto obtain the desired end concentration. Finally, the suspension wastransferred in anaerobic sterile vials and stored at −20° C.

Live bacteria in fresh and frozen suspensions were enumerated byperforming 1:10 serial dilutions of the suspension in anaerobicphosphate-carbonate buffer. 100 μL of each dilution was then plated intriplicates on pre-reduced agar YCFA petri dishes. Colonies were countedafter 5 days of incubation at 37° C. in anaerobic jars.

Mice

A set of 10-week-old C57BL/6J mice (48 mice, n=12/group) (Janvier Labs,France) were housed in SPF (specific pathogen free) environment bygroups of 2 mice/cage, with free access to food and water. The mice werefed a control diet (CT) or a high-fat diet (HFD). A group of mice wastreated with an oral administration of daily prepared fresh cultures ofD. welbionis J115^(T) by oral gavage at the dose 5·10⁹ bacteria/0.2 mL(corresponding to 2·10⁹ cfu/0.2 mL) suspended in sterile anaerobicsolution of trehalose 15% in phosphate-carbonate buffer saline (HFD J115fresh). Another group of mice was treated with frozen solution of D.welbionis J115^(T) by oral gavage at the dose 5·10⁹ bacteria/0.2 mL(corresponding to 3.5·10⁸ cfu/mL) suspended in sterile anaerobicsolution of trehalose 15% in phosphate-carbonate buffer saline (HFD J115frozen). HFD and control groups were treated with an oral gavage of anequivalent volume of trehalose 15% in phosphate-carbonate buffer saline(Control and HFD). Treatment continued for 13 weeks. Mice wereeuthanized after a 6 h fasting period.

Food and water intake were recorded once a week. Body composition wasassessed by using 7.5 MHz time domain-nuclear magnetic resonance(TD-NMR) (LF50 minispec, Bruker, Rheinstetten, Germany).

Intestinal Energy Absorption

Six weeks after the beginning of the experiment, the feces of twoseven-days periods were collected. During the same time the food intakewas monitored. The feces were dried at 60° C. during 2 hours andweighted. Total energy of the diet and the feces was determined by bombcalorimetry (C1, IKA, USA). The net intestinal absorption is calculatedbased on the ingested and excreted energy and represented the proportionof ingested energy that was not recovered in feces output.

Results

To decipher if dosage and/or viability of Dysosmobacter welbionisJ115^(T) influences the protection against HFD-induced obesity and themetabolic alterations and if frozen bacteria are as active asdaily-cultivated bacteria, 5·10⁹ fresh and frozen bacteria wereadministered per day to HFD-fed mice for 13 weeks. Enumeration of cfubefore and after preparation for daily fresh administration indicatedthat 40% of the bacteria survived, thus a 5·10⁹ cell dose correspond to2·10⁹ live, that is to say cultivable, bacteria (FIG. 18). Conversely,enumeration before and after preparation of frozen bacteria indicatedthat only 7% of the bacteria survived, thus a 5·10⁹ cell dosecorresponds to 3.5 10⁸ cultivable bacteria (FIG. 18).

Results show that supplementation with 5·10⁹ freshly prepared bacteriaper day decreased HFD-induced weight gain by and 24.7 and 26.8% (i.e.,3.5 and 4.9 g) after 8 and 13 weeks of treatment, respectively (FIG. 19and FIG. 20). Similarly, supplementation with 5·10⁹ frozen bacteria perday decreased HFD-induced weight gain by 22.8 and 33.3% (i.e., 3.1 and5.8 g) after 8 and 13 weeks of treatment, respectively (FIG. 19 and FIG.20). The extent of the effect of the fresh and frozen preparation issimilar after 8 weeks of treatment and slightly higher for the frozenpreparation, although not statistically significant, at the end of thestudy (13 weeks). As D. welbionis J115^(T) preparations with the sametotal amount but different viability levels exert the same physiologicaleffect on mice, it appears that the viability is not a necessary featureto obtain anti-obesity action. In other words, dead, non-cultivablecells of D. welbionis J115^(T) have the same beneficial properties thanlive, cultivable, cells of D. welbionis J115^(T). Fresh and frozen D.welbionis J115^(T) administration significantly decreased food intakeand thus total energy intake by 7.9 and 9.0% over the course of theexperiment (FIG. 21). To verify that this decreased food intake indeedresulted in a decrease in the energy available for host metabolism, thefeces of two periods of seven days were collected and the total energycontained in those feces was measured by bomb calorimetry. Energy outputin the feces was not affected by fresh or frozen D. welbionisadministration (FIG. 22 and FIG. 23), however, the quantity of caloriesabsorbed remained 8.2 and 9.5% lower than in HFD mice (FIG. 24).Furthermore, the ratio of weight gain over food intake was 19.5 and25.6% lower in fresh and frozen D. welbionis supplemented mice than inHFD mice (FIG. 25). This indicates that D. welbionis decreased theability to convert food energy into body mass. Taken together, thisresult show that D. welbionis prevents high fat diet induced weight bytwo complementary mechanisms, that is a decreased food intake and alower ability to store the absorbed energy.

Conclusion

These results confirm the observations made in example 5 that theadministration of J115 lead to the amelioration of several deleteriousconsequences associated with an high fat diet: a decrease of the gain ofweight, a decrease in food intake, a decrease of the gain of weight perfood intake and a decrease in energy absorption in the intestine. Theseobservations point to the beneficial effect of the administration ofJ115 for the treatment of metabolic diseases (notably obesity) andfeeding disorders. These effects are confirmed for a daily dose of 5·10⁹J115 cells/mice and are observed over a range of 3.5·10⁸ to 2·10⁹viable/cultivable J115 cells/mice/day. Notably the administration offrozen J115 cells, comprising 7% viable/cultivable cells is as efficientas not frozen preparation comprising 40% viable/cultivable cells. Thissuggest that not viable J115 cells are also able to ameliorate health inrespect to the several deleterious consequences associated with an highfat diet: a decrease of the gain of weight, a decrease in food intake, adecrease of the gain of weight per food intake and a decrease in energyabsorption in the intestine.

Example 8: Effect of D. Welbionis Administration on Adipose TissuesMaterial and Methods Dysosmobacter Welbionis J115^(T) Cultivation andEnumeration

Cf. corresponding section in Example 7.

Mice

Cf. corresponding section in Example 7.

Tissue Sampling

The animals were anesthetized with isoflurane (Forene®, Abbott,Queenborough, Kent, England) before exsanguination and tissue sampling,then mice were killed by cervical dislocation. The intestinal segments(duodenum, jejunum, ileum, cecum and colon) were dissected, immersed inliquid nitrogen, and stored at −80° C., for further analysis.

Adipocytes Diameter in Subcutaneous and Mesenteric Adipose Tissues

Paraffin sections of 5 μm were stained with hematoxylin and eosin Imageswere obtained using a SCN400 slide scanner and Digital Image Hubsoftware (Leica Biosystems, Wetzlar, Germany) Adipocytes size anddistribution were calculated from five fields per sample using Fiji andAdiposoft softwares.

Results

Obesity is associated with fat deposits expansion and disturbed adiposetissue function characterized by adipocyte hypertrophy, impairedlipolysis and pro-inflammatory phenotype, which contributes to insulinresistance and ectopic fat deposit. Results show that supplementationwith fresh and frozen 5·10⁹ D. welbionis J115^(T) cells per daydecreased by 20.0 and 24.2% (i.e., 3.1 and 3.8 grams) high-fat inducedadipose tissue weight gain (FIG. 26). This corresponds to significantdecreases of the weight of mesenteric, subcutaneous (inguinal) andepididymal fat deposits (FIG. 27). As adipocytes hypertrophy ischaracteristic of adipocytes dysfunction, adipocyte diameter wasmeasured in the subcutaneous (SCAT) and mesenteric (MAT) adipose tissueby histology and image analysis. Fresh and frozen D. welbionis J115^(T)treatment significantly increased the proportion of small adipocytes(diameter inferior to 50 μm) and decreased the proportion of largeadipocytes (diameter above 70 μm) in SCAT (FIG. 28 and FIG. 29).Visceral adipose tissue dysfunction is particularly associated tometabolic alteration, thus adipocytes size and distribution in MAT wasalso measured. Supplementation with fresh and frozen D. welbionisJ115^(T) cells per day significantly increased the proportion of smalladipocytes and decreased the proportion of large adipocytes in MAT (FIG.30 and FIG. 31). Strikingly, D. welbionis J115^(T) treatment completelyabolished HFD-induced MAT adipocytes hypertrophy as the adipocyte sizedistribution was identical in control diet fed mice and in HFD fed micetreated with fresh or frozen D. welbionis J115^(T).

Conclusion

These results indicate that the administration of J115 lead to theamelioration of several deleterious consequences associated with an highfat diet: a decrease the weight of adipose tissue (including mesenteric,subcutaneous (inguinal) and epididymal fat deposits), a decrease ofadipocyte hypertrophy (including in SCAT and MAT). These observationspoint to the beneficial effect of the administration of J115 for thetreatment of metabolic diseases (notably obesity, metabolic syndrome,hypertension, ectopic fat deposition, type 2 diabetes and dyslipidemia).These effects are observed for a daily dose of 5·10⁹ J115 cells/mice andare observed over a range of 3.5·10⁸ to 2·10⁹ viable/cultivable J115cells/mice/day. Notably the administration of frozen J115 cells,comprising 7% viable/cultivable cells is as efficient as not frozenpreparation comprising 40% viable/cultivable cells. This suggest thatnot viable J115 cells are also able to ameliorate health in respect tothe several deleterious consequences associated with an high fat diet: adecrease the weight of adipose tissue (including mesenteric,subcutaneous (inguinal) and epididymal fat deposits), a decrease ofadipocyte hypertrophy (including in SCAT and MAT).

Example 9: Effect of D. Welbionis Administration on Metabolic HealthMaterial and Methods Dysosmobacter Welbionis J115^(T) Cultivation andEnumeration

Cf. corresponding section in Example 7.

Mice

Cf. corresponding section in Example 7.

Oral Glucose Tolerance Test (OGTT)

After 13 weeks of treatment, six-hours-fasted mice were treated with anoral gavage glucose load (2 g glucose per kg body weight). Blood glucosewas measured 30 minutes and at time 0 just before oral glucose load andthen 15, 30, 60, 90 and 120 min after oral glucose load. Blood glucosewas determined with a glucose meter (Accu Check, Roche, Switzerland) onblood samples collected from the tip of the tail vein. Plasma insulinconcentration was determined using an ELISA kit (Mercodia, Uppsala,Sweden) according to the manufacturer's instructions.

Fasting Leptin Concentration

Circulating leptin concentration was determined using a multipleximmunoassay kit (Mouse diabetes assay, Bio-Plex Pro, Bio-Rad, Belgium)and measured using Luminex technology (Bioplex, Bio-Rad, Belgium).

Results

In order to better characterize the impact of D. welbionis J115^(T)treatment on glucose metabolism and metabolic health, oral glucosetolerance test were performed after 12 weeks of HFD and fresh and frozenD. welbionis J115^(T) supplementation. Fasting plasma glucose wassimilar in treated and non-treated mice, as 15 min after glucose load.However, the decrease of plasma glucose was faster in mice supplementedwith fresh and frozen D. welbionis J115^(T) (FIG. 32). In addition,plasma insulin levels tended to be lower before and 15 min after theglucose load (FIG. 33) despite similar plasma glucose levels, indicatingthat insulin sensitivity was likely improved by fresh and frozen D.welbionis J115^(T). Coherently with a better metabolic health, fastingplasma leptin levels were significantly lowered by fresh and frozen D.welbionis J115^(T) (FIG. 34).

Conclusion

These results indicate that the administration of J115 lead to theamelioration of several deleterious consequences associated with an highfat diet: a decrease of glucose tolerance, of insulin sensitivity and adecrease of fasting plasma leptin level. These observations point to thebeneficial effect of the administration of J115 for the treatment ofmetabolic diseases (notably obesity, insulin resistance, glucoseintolerance, hyperglycemia, metabolic syndrome, type 2 diabetes, type 1diabetes, dyslipidemia, altered endogenous glucose production). Theseeffects are observed for a daily dose of 5·10⁹ J115 cells/mice and areobserved over a range of 3.5·10⁸ to 2·10⁹ viable/cultivable J115cells/mice/day. Notably the administration of frozen J115 cells,comprising 7% viable/cultivable cells is as efficient as not frozenpreparation comprising 40% viable/cultivable cells. This suggest thatnot viable J115 cells are also able to ameliorate health in respect tothe several deleterious consequences associated with an high fat diet: adecrease of glucose tolerance, of insulin sensitivity and a decrease offasting plasma leptin level.

Example 10: Effect of D. Welbionis Administration on Brown andSubcutaneous Adipose Tissue Material and Methods Dysosmobacter WelbionisJ115^(T) Cultivation and Enumeration

Cf. corresponding section in Example 7.

Mice

Cf. corresponding section in Example 7. For experiment presented inFIGS. 36 and 37 a set of 10-week-old C57BL/6J mice (14 mice, n=7/group)(Janvier Labs, France) were housed individually, with free access tofood and water. The mice were fed a high-fat diet (HFD). A group of micewas treated with frozen solution of D. welbionis J115^(T) by oral gavageat the dose 5·10⁹ bacteria/0.2 mL (corresponding to 3.5·10⁸ cfu/mL)suspended in sterile anaerobic solution of trehalose 15% inphosphate-carbonate buffer saline (HFD J115 frozen). HFD groups wastreated with an oral gavage of an equivalent volume of trehalose 15% inphosphate-carbonate buffer saline (HFD). Treatment continued for 3weeks. On the last day of the experiment, the temperature of the micewas measured using a rodent rectal thermometer. Mice were euthanized inthe morning with no fasting period.

Tissue Sampling

Cf. corresponding section in Example 8.

RNA Preparation, Real-Time qPCR and RNAseq Analysis

RNA preparation and qPCR analyses were realized as described incorresponding section in Example 5. The primer presented in table 8bellow were used.

TABLE 8 Nucleotide sequence of the primer pairs used forthe measurement of mRNAs expression level by qPCR. SEQ ID PrimersSequence NO: F4/80 Forward TGACAACCAGACGGCTTGTG 11 ReverseGCAGGCGAGGAAAAGATAGTGT 12 ColA1 Forward CCTCAGGGTATTGCTGGACAAC 13Reverse ACCACTTGATCCAGAAGGACCTT 14

For RNAseq analysis, the integrity of RNA from brown adipose tissue(BAT) and subcutaneous adipose tissue (SCAT) was determined using withthe Agilent bioanalyzer 2100 system with the RNA 6000 Nano LabChip kit.BAT samples with an RNA integrity number inferior to 8 on a scaleranging from 0 to 10 were eliminated, as well as SCAT samples with anRNA integrity number inferior to 6.5. Then, samples were pooled (HFDpool and HFD-J115 pool) to an end concentration of 50 ng/μL. The sampleshave been sequenced by Eurofins Genomics, which consisted inpurification of poly-A containing mRNA molecules, then mRNAfragmentation, random primed cDNA synthesis (strand specific), adapterligation and adapter specific PCR amplification and finally paired-endIllumina sequencing with a read length of 2×150 bp. 80 to 90 millionread pairs were obtained and analyzed on Galaxy server using RNA-STARand htseq modules.

Results

D. welbionis J115^(T) decreased the ability to convert food energy intobody mass. This phenomenon can be the consequence of increased energyexpenditure. Brown adipose tissue (BAT) is a fat deposit that isparticularly metabolically active. Indeed, it is the major site ofnon-shivering thermogenesis, thereby controlling whole-body temperature,energy expenditure and body fat. Results show that supplementation with5·10⁹ freshly prepared and frozen bacteria per day decreased the BATweight by 24.7 and 26.8% (i.e., 61.6 and 56.4 mg) after 13 weeks oftreatment, respectively (FIG. 35). In order to confirm the effect of D.welbionis J115^(T) on body temperature and energy expenditure throughthe modulation of BAT metabolism, a set of 10-week-old HFD-fed C57BL/6Jmice were subjected to a 3-weeks treatment with either vehicle or 5·10⁹frozen cells of D. welbionis J115^(T) (14 mice, n=7/group, experiment3). The body temperature of mice treated with frozen D. welbionisJ115^(T) was 0.27° C. higher than the body temperature of control micefed a HFD and treated with vehicle (39.0° C. vs 38.73° C., FIG. 36). Inaddition, the BAT weight of those mice was negatively correlated withthe body temperature (FIG. 37). Fibrosis and inflammation areincreasingly appreciated as triggers of adipose tissue dysfunction. Todetermine if the adipose tissue metabolism improvement observed in micetreated with D. welbionis J115^(T) derives from lower inflammation andfibrosis levels, RNAseq analysis was performed in the BAT and SCAT ofHFD and the HFD-J115-frozen groups. The 22 genes related toextra-cellular matrix, that is too abundant in fibrosis, weredown-regulated in the BAT and SCAT of mice treated with frozen D.welbionis J115^(T) (Table 9).

TABLE 9 RNAseq analysis in BAT and SCAT, representing the relativeexpression of genes related to fibrosis and extra-cellular matrix ofmice treated by daily oral gavage with frozen suspensions of 5.10⁹ cellsof D. welbionis J115^(T) and fed a HF-diet relative to mice fed ahigh-fat diet (HFD) and treated by daily oral gavage with an equivalentvolume of sterile trehalose 15% in anaerobic PBS-carbonate buffer salinefor 13-weeks (n = 12/group). Fold-change HFD-J115-frozen/HFD Gene BATSCAT Col3a1 0.53 0.6 Col1a2 0.45 0.6 Col6a1 0.72 0.65 Col1a1 0.47 0.67Col5a2 0.49 0.62 Anxa1 0.73 0.58 Serpinf1 0.65 0.57 Cilp 0.76 0.54Adamts4 0.65 0.94 Ntn1 0.62 0.86 Ltbp3 1 1 Col6a2 0.24 0.67 Ctsc_2 0.620.56 Mmp14 0.85 0.59 Nid2 0.75 0.78 Lum 0.8 0.45 S100a6 0.4 0.52 Thbs20.18 0.38 Col10a1 0.86 1 Myoc 0.43 0.68 Cela1 1 0.48 Adamts12 1 0.39

Accordingly, 50 genes related to inflammatory response gene ontologywere all but one down-regulated in the BAT of mice treated with D.welbionis J115^(T) in comparison with HFD-fed mice, demonstrating thatD. welbionis J115^(T) protects adipose tissue against HFD-inducedinflammation and fibrosis (FIG. 38). The relative mRNA expression of twogenes related to inflammation (F4/80, a marker of macrophagesinfiltration) and fibrosis (collagen A1, a constituent of extra-cellularmatrix) was also measured by qPCR and it was confirmed that these geneswere up-regulated by HFD in comparison with ND and normalized by bothfresh and frozen D. welbionis J115^(T) (FIG. 39 and FIG. 40).

Conclusion

These results indicate that the administration of J115 lead to theamelioration of several deleterious consequences associated with an highfat diet: a decrease of the weight of brown adipose tissues, a decreaseof fibrosis in the brown and subcutaneous adipose tissues and a decreaseof inflammation in the brown adipose tissue. These observations point tothe beneficial effect of the administration of J115 for the treatment ofmetabolic diseases (notably obesity, adipose tissues inflammation,adipose tissues fibrosis and abnormal fat accumulation, alteredlipolysis, high-fat storage). These effects are observed for a dailydose of 5·10⁹ J115 cells/mice and are observed over a range of 3.5·10⁸to 2·10⁹ viable/cultivable J115 cells/mice/day. Notably theadministration of frozen J115 cells, comprising 7% viable/cultivablecells is as efficient as not frozen preparation comprising 40%viable/cultivable cells. This suggest that not viable J115 cells arealso able to ameliorate health in respect to the several deleteriousconsequences associated with an high fat diet: a decrease of the weightof brown adipose tissues, a decrease of fibrosis in the brown andsubcutaneous adipose tissues and a decrease of inflammation in the brownadipose tissue.

Example 11: Effect of D. Welbionis Strain J115^(T) Administration onTLR2 Signaling and the Intestinal Barrier Material and MethodsDysosmobacter Welbionis J115^(T) Cultivation and Enumeration

Cf. corresponding section in Example 7.

Mice

Cf. corresponding section in Example 7.

Tissue Sampling

Cf. corresponding section in Example 8.

RNA Preparation, Real-Time qPCR and RNAseq Analysis

RNA preparation, qPCR analyses and were realized as described incorresponding section in Examples 5 and 10. The primer presented inTable 10 below were used for dPCR analysis.

TABLE 10 Nucleotide sequence of the primer pairs used forthe measurement of mRNAs expression level by qPCR. SEQ ID PrimersSequence NO: DEFA Forward GGTGATCATGAGACCCCAGCATCAGT 15 ReverseAAGAGACTAAAAGTGAGGAGCAGC 16In Vitro Culture and Stimulation of Human HEK-Blue hTLR2 Cell Lines.

For the immune receptor stimulation analysis HEK-Blue hTLR2 cell line(Invivogen, CA, USA) was used. Stimulation of the hTLR2 with its ligandsactivates NFκB, which induces the production of secreted embryonicalkaline phosphatase (SEAP), the levels of which were measured using theQUANTI-Blue colorimetric enzyme assay and a spectrophotometer(Spectramax, Molecular Devices, CA, USA). HEK-Blue hTLR2 cell line wasgrown and cultured up to 70-80% of confluency using as a maintenancemedium DMEM supplemented with 4.5 g/l D-glucose, 50 U/mL penicillin, 50μg/mL streptomycin, 100 μg/mL Normocin, 2 mM L-glutamine and 10% (v/v)of heat-inactivated FBS Immune response experiment was carried out byseeding HEK-hTLR2 cells in flat-bottom 96-well plates (50 000 cells per200 μL well) and stimulating them by addition of 20 μl bacterialsuspensions or Pam3CSK4 (synthetic triacylated lipopeptide, Invivogen,CA, USA) as positive control. The 96-well plates were incubated for 24 hat 37° C. in a 5% CO₂/95% air atmosphere. SEAP secretion was detected bymeasuring the OD600 at 1 h after addition of 180 μL of QUANTI-Blue(Invivogen) to 20 μL of induced HEK-Blue hTLR2 supernatant.

Results

Stimulation of toll-like receptor 2 (TLR2) in the intestine is known tostrengthen barrier function and improve metabolic health. Results showthat frozen D. welbionis J115^(T) specifically activated cellsexpressing TLR2 in a dose-dependent manner (FIG. 41). The EC50 is thehalf-maximal effective concentration and refers to the concentration ofbacteria that induces a response halfway between the baseline andmaximum after 2 h of exposure. In the case of D. welbionis J115^(T),this EC50 is 3.9 10⁵ cells per mL, which is a relatively lowconcentration and indicates that D. welbionis J115^(T) is a strongpotent activator of TLR2 (FIG. 42). TLR2 signaling is known to beassociated to barrier function strengthening. Indeed, RNAseq analysis inthe jejunum showed that the expression of the genes encodingantimicrobial peptides and tight junction proteins was upregulated inthe jejunum of mice treated with D. welbionis J115^(T) (Table 11).

TABLE 11 RNAseq analysis in the jejunum, representing the relativeexpression of genes related to tight junction proteins and antimicrobialpeptides of mice treated by daily oral gavage with frozen suspensions of5.10⁹ cells of D. welbionis J115^(T) and fed a HF-diet relative to micefed a high-fat diet (HFD) and treated by daily oral gavage with anequivalent volume of sterile trehalose 15% in anaerobic PBS-carbonatebuffer saline for 13-weeks (n = 12/group). Gene name Fold-changeHFD-J115-frozen/HFD Cldn15 1.52 Cldn23 1.08 Cldn3 1.3 Cldn4 2.4 Cldn71.08 Ocln 1.06 Muc2 1.36 Defa17 1.46 Defa2 1 Defa20 1 Defa21 2.5 Defa222.06 Defa24 1.56 Defa30 1.33 Defa32 1 Defa34 2.5 Defa5 1.55 Reg1 1.54Reg3a 1.18 Reg3b 1.31 Reg3g 1.73

The relative mRNA expression of defensin a was also measured by qPCR andit confirmed that this gene is up-regulated by both fresh and frozen D.welbionis J115^(T) (FIG. 43).

Conclusion

These results indicate that the administration of J115 lead to theamelioration of several deleterious consequences associated with an highfat diet: an increase in the expression of tight junction related genesand an increase in the expression of antimicrobial peptides.Furthermore, J115 is a strong activator of TLR2. The activation of TLR2promotes anti-inflammatory pathways reinforce the intestinal barrier(Oppong et al., Infect Immun 2013 February; 81(2):478-86). Theseobservations point to a beneficial effect of the administration of J115for the treatment of diseases associated with a dysfunctional intestinalbarrier notably intestinal inflammation, food allergies, celiac disease,colitis, ulcers, infection, hepatic diseases, steatohepatitis. Theseeffects are observed for a daily dose of 5·10⁹ J115 cells/mice and areobserved over a range of 3.5·10⁸ to 2·10⁹ viable/cultivable J115cells/mice/day. Notably the administration of frozen J115 cells,comprising 7% viable/cultivable cells is as efficient as not frozenpreparation comprising 40% viable/cultivable cells. This suggests thatnot viable J115 cells are also able to ameliorate health in respect tothe several deleterious consequences associated with an high fat diet:an increase in the expression of tight junction related genes, anincrease in the expression of antimicrobial peptides and an efficientactivation of TLR2 signaling.

Example 12: The Proportion of D. Welbionis in the Human IntestinalMicrobiota Correlates Negatively with Weight Material and Methods

Dysosmobacter Spp Quantification in Stools by qPCR

Genomic DNA was extracted from human stools using the QIAamp DNA StoolMini Kit (Qiagen, Germany), including a bead-beating step. DNAconcentration was determined and purity (A260/A280) was checked using aNanoDrop2000 (Thermo Fisher Scientific, USA). Samples were diluted to anend concentration of 10 and 0.1 ng/μL in TE buffer pH 8. A standardcurve was included on each plate by diluting genomic DNA from pureculture. Cell counts were determined by plating and expressed as cfubefore DNA extraction. Dysosmobacter spp quantity in stools wasexpressed as percentage of total bacteria. qPCR was performed asdescribed in example 5 with primers specific for Dysosmobacter speciesand primer allowing amplification from all bacteria (Table 12).

TABLE 12 Nucleotide sequence of the primer pairs used forthe Dysosmobacter spp proportion in human stools. SEQ ID PrimersSequence NO: Dysosmo- Forward ATGACGCATGACGCATGACC 17 bacter spp ReverseCCAGCGATAAAATCTTTGACATGCC 18 Total Forward ACTCCTACGGGAGGCAGCAG 19Bacteria Reverse ATTACCGCGGCTGCTGG 20

Results

The relative abundance of the genus Dysosmobacter in the fecalmicrobiota of 62 individuals with a body mass index (BMI) ranging from18.0 to 50.7 kg·m⁻² was measured by qPCR. Results show that the log ofDysosmobacter spp relative abundance was negatively correlated with BMIin this cohort with a p value below 0.0001 (FIG. 44). In conjunctionwith preclinical data obtained with mouse models, this result confirmsthat bacteria belonging to the Dysosmobacter genus and D. welbionisJ115^(T) in particular protect against obesity and contributes toslenderness and metabolic health. In subjects with a BMI bellow 25, theproportion of Dysosmobacter was 2.45%. In subjects with a BMI rangingfrom 25 to 30, the proportion of Dysosmobacter was 1.5%. In subjectswith a BMI ranging from 30 to 35, the proportion of Dysosmobacter was0.85%. In subjects with a BMI ranging from 35 to 40, the proportion ofDysosmobacter was 0.5%.

Conclusion

These observations suggest a positive contribution of the presence ofbacteria of the Dysosmobacter genus and D. welbionis J115^(T) inparticular in the human intestinal microbiota. The negative correlationobserved is not limited to obese patient (with a BMI superior to 30)with it is also includes healthy subjects (with a BMI inferior to 30 andwith a BMI inferior to 25). These observations again point to thebeneficial effect of the administration of J115 for the treatment ofmetabolic diseases (notably obesity). The correlation being made usinghealthy subjects also point to the beneficial effect of theadministration of J115 in healthy subject to improve well-being and forcosmetic purpose, notably to promote weight loss and/or prevent weightgain.

1-15. (canceled)
 16. An isolated bacterium belonging to the genusDysosmobacter and/or a variant thereof and/or fragments thereof.
 17. Theisolated bacterium according to claim 16, wherein said bacterium belongsto the species Dysosmobacter welbionis and/or a variant thereof.
 18. Theisolated bacterium according to claim 16, wherein the nucleotidesequence of the 16S rRNA gene of said isolated bacterium has at leastabout 90% identity with SEQ ID NO:
 1. 19. The isolated bacteriumaccording to claim 16, wherein said bacterium is able to fermentmyo-inositol.
 20. The isolated bacterium according to claim 16, whereinsaid bacterium is the strain J115, deposited at the BCCM/LMG on Mar. 14,2018 as LMG P-30603, and/or a variant thereof.
 21. The isolatedbacterium according to claim 16, wherein said bacterium is pasteurized.22. The isolated bacterium according to claim 16, wherein said bacteriumis frozen.
 23. A composition comprising an isolated bacterium belongingto the genus Dysosmobacter and/or a variant thereof and/or fragmentsthereof.
 24. The composition according to claim 23, wherein saidcomposition is a pharmaceutical composition comprising at least onepharmaceutically acceptable excipient.
 25. The composition according toclaim 23, wherein said composition is a nutraceutical compositioncomprising at least one nutraceutically acceptable excipient.
 26. Thecomposition according to claim 23, wherein said composition is acosmetic composition comprising at least one cosmetically acceptableexcipient.
 27. A method for treating a disorder in a subject in needthereof, comprising administering to said subject at least one isolatedbacterium belonging to the genus Dysosmobacter and/or a variant thereofand/or fragments thereof.
 28. The method according to claim 27, whereinsaid disorder is a disorder related to the gastrointestinal microbiota.29. The method according to claim 27, wherein said disorder is ametabolic disease.
 30. The method according to claim 27, wherein saiddisorder is selected from the list comprising obesity, metabolicsyndrome, insulin-deficiency or insulin-resistance related disorders,Diabetes Mellitus, glucose intolerance, abnormal lipid metabolism,hyperglycemia, dyslipidemia, high cholesterol, elevated LDL-cholesterol,decreased HDL-cholesterol and elevated triglycerides.
 31. A method forpromoting weight loss, decreasing food intake, increasing muscle mass,decreasing fat mass, increasing satiety, and/or decreasing weight gainassociated with food intake in a subject, comprising administering tosaid subject at least one isolated bacterium belonging to the genusDysosmobacter and/or a variant thereof and/or fragments thereof.